Continuous directed evolution

The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

1. A method of phage-assisted continuous evolution, comprising

(a) contacting a population of bacterial host cells in a culture medium with a population of M13 phages comprising a gene of interest to be evolved and lacking a functional pIII gene required for the generation of infectious phage particles, wherein

(1) the M13 phages allow for expression of the gene of interest in the host cells,

(2) the host cells are suitable host cells for M13 phage infection, replication, and packaging, wherein the M13 phage comprises all phage genes required for the generation of phage particles, except a full length pIII gene; and

(3) the host cells comprise an expression construct encoding the pIII protein, wherein expression of the pIII gene is dependent on a function of a gene product of the gene of interest;

(b) incubating the population of host cells under conditions allowing for the mutation of the gene of interest, the production of infectious M13 phage, and the infection of host cells with M13 phage, wherein infected cells are removed from the population of host cells, and wherein the population of host cells is replenished with fresh host cells that are not infected by M13 phage, wherein the function of the gene of interest is a desired function, wherein desired functional library members induce production of pIII and release M13 progeny into the culture medium capable of infecting new host cells, wherein undesired non-functional library members do not produce pIII and release only non-infectious M13 progeny into the culture medium;

(c) isolating a mutated M13 phage replication product encoding an evolved protein from the population of host cells, wherein the host cells comprise an expression construct encoding a dominant-negative pIII protein (pIII-neg), wherein the pIII-neg protein is driven by a promoter the activity of which depends on an undesired function of a gene product encoded by the gene of interest.

2. The method of claim 1, wherein the phage genes required for the generation of phage particles are pI, pII, pIV, pV, pVI, pVII, pVIII, pIX, and/or pX.

3. The method of claim 1, wherein the host cells comprise an accessory plasmid.

4. The method of claim 3, wherein the accessory plasmid comprises an expression construct encoding the pIII protein.

5. The method of claim 3, wherein the cell comprises the accessory plasmid and together, the phage genome and the accessory plasmid comprise all genes required for the generation of an infectious phage.

6. The method of claim 3, wherein the cell comprises a helper phage and an accessory plasmid and together, the helper plasmid and the accessory plasmid comprise all genes required for the generation of an infectious phage in the presence of a phage genome.

7. The method of claim 1, wherein the pIII expression construct comprises a conditional promoter regulating the expression of the pIII gene, wherein the activity of the conditional promoter depends on a function of a gene product encoded by the gene of interest.

8. The method of claim 7, wherein the function of the gene of interest is a binding activity or an enzymatic activity.

9. The method of claim 8, wherein the enzymatic activity is a polymerase activity, a recombinase activity, a phospho-transferase activity, a kinase activity, a phosphatase activity, or a protease activity.

10. The method of claim 9, wherein the expression of the pIII protein is driven by a promoter comprising a cI binding site, and wherein the cell expresses a cI protein comprising a desired target protease cleavage site.

11. The method of claim 9, wherein the expression of the pIII-neg protein is driven by a promoter comprising a cI-neg binding site, and wherein the cell expresses a cI-neg protein comprising an undesired target protease cleavage site.

12. The method of claim 1, wherein the host cells further comprise a mutagenesis plasmid.

13. The method of claim 11, wherein the mutagenesis plasmids comprises a gene expression cassette encoding a mutagenesis-promoting gene product.

14. The method of claim 12, wherein the expression cassette comprises a nucleic acid encoding a gene involved in the SOS stress response.

15. The method of claim 11, wherein the expression cassette encoding a mutagenesis-promoting gene product comprises a conditional promoter, the activity of which depends on the presence of an inducer.

16. The method of claim 12, further comprising contacting the host cells with an inducer that induces the expression of the mutagenesis-promoting genes in the host cells.

17. The method of claim 1, wherein the host cells are E. coli cells.

18. The method of claim 1, wherein the cells are exposed to a mutagen.

19. The method of claim 1, wherein the expression construct encoding the pIII protein and the expression construct encoding the pIII-neg protein are situated on the same plasmid.

20. The method of claim 14, wherein the gene involved in the SOS stress response is UmuC, UmuD′, and/or RecA.

This application is a national stage filing under 35 U.S.C. §371 of international PCT application, PCT/US2011/066747, filed Dec. 22, 2011, which claims priority under 35 U.S.C. §119(e) to U.S. provisional patent application, U.S. Ser. No. 61/426,139, filed Dec. 22, 2010, entitled “Continuous Directed Evolution,” the entire contents of each of which are incorporated herein by reference.

This invention was made with government support under grant R01 GM065400 awarded by the National Institutes of Health. The government has certain rights in this invention.

Conventional directed evolution involves discrete cycles of mutagenesis, transformation or in vitro expression, screening or selection, and gene harvesting and manipulation. [1,2] In contrast, evolution in nature occurs in a continuous, asynchronous format in which mutation, selection, and replication occur simultaneously. Although successful evolution is strongly dependent on the total number of rounds performed, [3] the labor- and time-intensive nature of discrete directed evolution cycles limit many laboratory evolution efforts to a modest number of rounds.

In contrast, continuous directed evolution has the potential to dramatically enhance the effectiveness of directed evolution efforts by enabling an enormous number of rounds of evolution to take place in a single experiment with minimal researcher time or effort. In a landmark experiment, Joyce and co-workers engineered a ribozyme self-replication cycle in vitro and used this cycle to continuously evolve a ribozyme with RNA ligase activity. This system remains the only reported example of continuous directed evolution and unfortunately cannot be easily adapted to evolve other biomolecules: [4-7]

Continuous directed evolution minimally requires (i) continuous mutagenesis of the gene(s) of interest, and (ii) continuous selective replication of genes encoding molecules with a desired activity. Several groups have developed methods to achieve continuous or rapid non-continuous cycles of mutagenesis. [8-13] For example, Church and coworkers recently developed multiplex automated genome engineering (MAGE), a system capable of generating targeted diversity in E. coli through automated cycles of transformation and recombination. [14] While these advances are capable of very efficiently creating gene libraries, they have not been linked to a rapid and general continuous selection, and consequently have not enabled continuous directed evolution.

Laboratory evolution has generated many biomolecules, e.g., nucleic acids and proteins, with desired properties. However, conventional laboratory evolution strategies rely on discrete mutation-selection rounds, and a single round of directed evolution typically requires days or longer with frequent intervention by a researcher, making complex evolution processes impractical to perform in the laboratory.

The present invention provides a platform that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in a host cell, for example, in an E. coli cell. Some aspects of this invention provide a method of evolving a gene of interest by linking an activity of a molecule encoded by the gene of interest to the transfer of the gene of interest from cell to cell. In some embodiments, the platform includes a means of transferring a gene encoding a molecule of interest, for example, a protein or an RNA, from cell to cell. For example, the platform may include a phage genome, comprising a gene of interest, also referred to herein as a gene to be evolved, which replicates and mutates in a flow of host cells. The desired function of the gene of interest drives expression of a gene in the host cells that is essential for transfer of the gene from one cell to another, thus providing a selective advantage for those vectors in which the gene of interest has acquired a relevant gain-of-function mutation. For example, in some embodiments, a gene encoding a phage protein required for the production of infectious phage particles under the control of a conditional promoter the activity of which depends on a gene product encoded by the gene of interest. A population of transfer vectors comprising a gene of interest can continuously evolve in a flow of host cells over multiple transfer cycles, for example, multiple viral life cycles, and simultaneously be selected for a desired function of the gene of interest, or a product thereof, by predicating a step in the transfer cycle on the desired function, or product, thus conferring a competitive advantage on such mutants in the vector population.

Methods and vectors for the transfer of a gene of interest from cell to cell are well known in the art and include, for example, infection of suitable host cells with a viral vector (e.g., bacterial cells with a bacteriophage vector, mammalian cells with a retroviral vector, e.g., a vesicular stomatitis virus vector or a lentiviral vector), transfer of plasmids via bacterial conjugation, or intentional lysis of a cell comprising a gene of interest and uptake of naked DNA by a nearby cell.

In some embodiments, the evolution process is continuous, while in others, it is semi-continuous or involves discrete steps. In some embodiments, the evolution process is autonomous, or runs with only minimal human intervention, for example, by employing a system of controllers regulating the flow of cells or the rate of gene transfer between cells, or a robotic system performing multiple discrete steps or cycles of an evolution process.

The invention provides systems, methods, apparatuses, materials, cell lines, vectors, viruses, and reagents for directed continuous evolution, in which evolving genes are transferred from host cell to host cell through a modified viral life cycle in a manner that is dependent on an activity of interest. Dozens of cycles of viral replication, mutation, and selection can occur in a single day of directed continuous evolution without human intervention.

For example, in some embodiments, the invention provides systems, methods, apparatuses, materials, cell lines, vectors, viruses, and reagents for phage-assisted continuous evolution (PACE), in which evolving genes are transferred from host cell to host cell through a modified phage life cycle in a manner that is dependent on an activity of interest. In some such embodiments, a phage vector is provided that comprises a gene of interest to be evolved and is deficient in a gene required for the generation of infectious phage particles. In some embodiments, the gene required for the generation of infectious phage particles is under the control of a conditional promoter the activity of which is dependent on a desired activity of a product of the gene of interest (e.g., a protein or nucleic acid). In some embodiments, the phage vector is contacted with a population of host cells under conditions allowing for mutation of the gene of interest. A mutation in a gene of interest that confers a gain of the desired activity provides a competitive advantage over phage vectors without such a mutation or with a loss-of-function mutation, thus providing a continuous selective pressure on the mutating phage. Since phage life cycles are generally short (e.g., in the range of 10-20 minutes), a plurality of phage life cycles can occur in a single day of directed continuous evolution without human intervention.

As an example of a PACE process, T7 RNA polymerase (RNAP), a polymerase with highly specific requirements as to its promoter recognition sequence, was evolved to recognize a distinct promoter and to initiate transcripts with nucleotides other than G. See Example section below. Each of the three evolved activities emerged in less than one week of continuous evolution, and in all three cases PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on the wild-type T7 promoter. By greatly accelerating laboratory evolution, PACE provides efficient solutions to intractable directed evolution problems.

This invention provides methods for continuous evolution of biomolecules, for example, of nucleic acids, of proteins, or of small molecules synthesized via a biosynthetic pathway. In some embodiments, a method is provided that includes the steps of (a) contacting a population of host cells with a population of viral vectors comprising a gene of interest, wherein the host cells are amenable to infection with the viral vector; (b) optionally, contacting the population of host cells with a mutagen; (c) incubating the population of host cells under conditions allowing for the production of viral particles, wherein cells are removed from the host cell population, and the population of host cells is replenished with fresh, uninfected host cells; and (d) isolating a mutated replication product of the viral vector, encoding an evolved gene of interest, from the population of host cells. In some embodiments, the expression of at least one viral gene required for the production of an infectious viral particle comprising a replication product of the viral vector is dependent on a function of a product, for example, a protein or nucleic acid, encoded by the gene of interest.

This invention also provides methods of phage-assisted continuous evolution (PACE) of a protein. In some embodiments, the method comprises providing a flow of host cells, for example, a flow of bacterial host cells through a lagoon, and a population of phage vectors encoding a gene of interest replicating in the flow of host cells under conditions allowing for replication and mutation of the phage genomes. In some embodiments, the host cells comprise a gene required for the generation of infectious phage under the control of a conditional promoter that is activated by a desired function of the gene of interest, thus conferring a selective advantage to phage genomes acquiring a gain-of function in the gene of interest. In some embodiments, the method includes contacting a population of bacterial host cells with a population of phage, for example, filamentous phages, such as M13 phages.

In some embodiments, a PACE method is provided in which a host cell population is contacted with a selection phage population, wherein the host cells comprise an expression construct in which expression of a gene required for the production of infectious phage is driven by a conditional promoter dependent on an activity of a gene product of the gene of interest, as described in more detail elsewhere herein. In some such embodiments, the selection phage comprises all remaining phage genes required for the generation of phage particles. For example, in some embodiments, a population of E. coli host cells comprising an expression construct, in which a nucleic acid encoding pIII is driven by a conditional promoter, is contacted with a population of M13 selection phage comprising a gene of interest and an origin of replication, and encoding pI, pII, and pIV-pX. In some such embodiments, the selection phage drives expression of the gene of interest from a phage promoter. In some such embodiments, the selection phage comprises all phage promoters of the wild-type phage, including the 3′-gIII promoter (see FIG. 16). In some embodiments, the selection phage comprises the last 180 bp of gIII, but not a full-length gIII coding sequence. In some embodiments, the host cells do not comprise a helper phage. One advantage of avoiding the use of a helper phage is that infection efficiency can be decreased in cells that express phage genes before infection. Accordingly, the use of a two-plasmid PACE vector system, comprising selection phage and accessory plasmid, is preferable, in some embodiments, over the use of a conventional, three-plasmid PACE vector system comprising a helper phage.

The invention also provides an apparatus or system for performing the continuous evolution methods provided herein. In some embodiments, the apparatus includes a lagoon holding an actively replicating population of phage vectors encoding a protein to be evolved, a population of phage host cells, an inflow, connected to a turbidostat holding fresh host cells, an outflow, and a controller regulating inflow and/or outflow. In some embodiments the flow rate through the lagoon is regulated so that the average time a host cell stays in the lagoon is longer than the phage life cycle, but shorter than the average time between cell divisions of the host cell. In some embodiments, the apparatus further comprises a vessel holding a mutagen, connected to an inflow of the lagoon. In some embodiments the apparatus further comprises a vessel holding an inducer of gene expression, connected to an inflow of the lagoon. In some embodiments, the host cell population comprises cells that harbor an accessory plasmid, as described in more detail elsewhere herein, but do not harbor a helper phage.

The invention further provides reagents for phage-assisted continuous evolution of biomolecules. In some embodiments, PACE vectors are provided, for example, a selection phage or phagemid, an accessory plasmid, a mutagenesis plasmid, and a helper plasmid. A selection phage or phagemid comprises a phage genome into which a gene of interest has been inserted, and in which a gene required for the generation of infectious phage particles is mutated, for example, by partial or complete deletion of the gene. In certain embodiments, the gene required for the generation of infectious phage particles is replaced, at least partially, with the gene of interest. The accessory plasmid includes the gene required for the generation of infectious phage under the control of a conditional promoter that is activated by a function of a gene product of the gene of interest and the helper phage or helper plasmid is provided that comprises an expression cassette for the expression of all genes required for the generation of phage particles that are not expressed from the selection phage or the accessory plasmid. In some embodiments, all genes required for the generation of infectious phage are expressed by the selection phage and the accessory plasmid, and a helper phage or plasmid is not required.

In some embodiments, a two-plasmid PACE vector system is provided, comprising a selection phage and an accessory plasmid, but no helper phage. In some such systems, the accessory plasmid comprises a gene encoding a protein required for the generation of infectious phage particles, and the selection phage provides all other phage genes required for phage particle generation, in addition to the gene of interest. Such a two-plasmid PACE vector system can be used by itself, or in combination with a mutagenesis plasmid as described elsewhere herein.

Host cells for PACE methods are also provided. In some embodiments, the host cells comprise an accessory plasmid with an expression construct in which the expression of a gene required for the generation of infectious phage, for example, pIII, is driven by a conditional promoter that is activated by a desired evolved function of the gene of interest. In some embodiments, the cells further comprise a mutagenesis plasmid for the expression of mutagenesis-inducing or -promoting genes, for example, genes of the bacterial SOS response or of proofreading-impaired DNA polymerases.

The invention also provides selection phages, viruses, or phagemids that include a gene of interest in their genome to be involved. In some embodiments, the selection phage is an M13 phage, the genome of which the pIII gene has been partially deleted and replaced with a gene of interest. In some embodiments, the selection phage comprises a wild-type M13 genome, comprising an origin of replication, all phage promoters and terminators comprised in the wild type genome (see FIG. 16 for reference), and a gene of interest inserted into the phage genome, but lacks a full-length coding region of a gene encoding a protein required for the generation of infectious phage particles. In some embodiments, the protein is pH′. In some such embodiments, the phage genome lacks a full-length pIII-encoding sequence, but comprises the 3′-fragment of gIII that comprises the promoter (see FIG. 16). In some embodiments, the 3′-fragment of gIII comprises the last 180 bases of gIII. In some embodiments, the gene of interest is a coding sequence that is cloned into the phage genome downstream of a wild type phage promoter. In some embodiments, the gene of interest does not comprise a transcriptional termination sequence, but uses a terminator of the phage genome. In some embodiments, the gene of interest is cloned into the phage genome to replace the gene or gene fragment encoding a protein required for the generation of infectious phage that is lacking in the phage genome. In some embodiments, the gene of interest is a coding nucleotide sequence cloned into an M13 selection phage downstream of the M13 promoter immediately downstream of the gVIII terminator, but upstream of the gIII 3′-promoter (see FIG. 16 for reference), in place of the nucleotide sequence encoding pIII in the wild-type M13.

The invention also provides evolved products of the inventive methodology. For example, in some embodiments, an evolved T7 RNA polymerase is provided that comprises a mutation conferring a modified substrate specificity and/or increased transcriptional activity to the evolved protein. Further, some aspects of this invention provide kits comprising reagents, vectors, cells, and/or apparatus for carrying out the methods provided herein. For example, in some embodiments, a kit for continuous directed evolution in a bacterial system is provided that includes a selection phage or phagemid; an accessory plasmid; optionally, a mutagenesis plasmid and/or a mutagen; and/or a host cell capable of producing infectious phage and amenable to phage infection. In some embodiments, a kit is provided that comprises a two-plasmid PACE vector system, as described in more detail elsewhere herein, for example, comprising a selection phage, an accessory plasmid, optionally, a mutagenesis plasmid, but no helper phage.

Other advantages, features, and uses of the invention will be apparent from the detailed description of certain non-limiting embodiments; the drawings, which are schematic and not intended to be drawn to scale; and the claims.

Continuous Evolution Concept

The term “continuous evolution,” as used herein, refers to an evolution procedure, in which a population of nucleic acids is subjected to multiple rounds of (a) replication, (b) mutation, and (c) selection to produce a desired evolved product, for example, a nucleic acid encoding a protein with a desired activity, wherein the multiple rounds can be performed without investigator interaction and wherein the processes under (a)-(c) can be carried out simultaneously. Typically, the evolution procedure is carried out in vitro, for example, using cells in culture as host cells. In general, a continuous evolution process provided herein relies on a system in which a gene of interest is provided in a nucleic acid vector that undergoes a life-cycle including replication in a host cell and transfer to another host cell, wherein a critical component of the life-cycle is deactivated and reactivation of the component is dependent upon a desired mutation in the gene of interest.

In some embodiments, a gene of interest is transferred from cell to cell in a manner dependent on the activity of the gene of interest. In some embodiments, the transfer vector is a virus infecting cells, for example, a bacteriophage, or a retroviral vector. In some embodiments, the viral vector is a phage vector infecting bacterial host cells. In some embodiments, the transfer vector is a retroviral vector, for example, a lentiviral vector or a vesicular stomatitis virus vector, infecting human or mouse cells. In some embodiments, the transfer vector is a conjugative plasmid transferred from a donor bacterial cell to a recipient bacterial cell.

In some embodiments, the nucleic acid vector comprising the gene of interest is a phage, a viral vector, or naked DNA (e.g., a mobilization plasmid). In some embodiments, transfer of the gene of interest from cell to cell is via infection, transfect ion, transduction, conjugation, or uptake of naked DNA, and efficiency of cell-to-cell transfer (e.g., transfer rate) is dependent on an activity of a product encoded by the gene of interest. For example, in some embodiments, the nucleic acid vector is a phage harboring the gene of interest and the efficiency of phage transfer (via infection) is dependent on an activity of the gene of interest in that a protein required for the generation of phage particles (e.g., pIII for M13 phage) is expressed in the host cells only in the presence of the desired activity of the gene of interest. In another example, the nucleic acid vector is a retroviral vector, for example, a lentiviral or vesicular stomatitis virus vector harboring the gene of interest, and the efficiency of viral transfer from cell to cell is dependent on an activity of the gene of interest in that a protein required for the generation of viral particles (e.g., an envelope protein, such as VSV-g) is expressed in the host cells only in the presence of the desired activity of the gene of interest. In another example, the nucleic acid vector is a DNA vector, for example, in the form of a mobilizable plasmid DNA, comprising the gene of interest, that is transferred between bacterial host cells via conjugation and the efficiency of conjugation-mediated transfer from cell to cell is dependent on an activity of the gene of interest in that a protein required for conjugation-mediated transfer (e.g., traA or traQ) is expressed in the host cells only in the presence of the desired activity of the gene of interest. Host cells contain F plasmid lacking one or both of those genes.

For example, some embodiments provide a continuous evolution system, in which a population of viral vectors comprising a gene of interest to be evolved replicates in a flow of host cells, e.g., a flow through a lagoon, wherein the viral vectors are deficient in a gene encoding a protein that is essential for the generation of infectious viral particles, and wherein that gene is comprised in the host cell under the control of a conditional promoter that can be activated by a gene product encoded by the gene of interest, or a mutated version thereof. In some embodiments, the activity of the conditional promoter depends on a desired function of a gene product encoded by the gene of interest. Viral vectors, in which the gene of interest has not acquired a mutation conferring the desired function, will not activate the conditional promoter, or only achieve minimal activation, while any mutation in the gene of interest that confers the desired mutation will result in activation of the conditional promoter. Since the conditional promoter controls an essential protein for the viral life cycle, activation of this promoter directly corresponds to an advantage in viral spread and replication for those vectors that have acquired an advantageous mutation.

The term “flow”, as used herein in the context of host cells, refers to a stream of host cells, wherein fresh host cells are being introduced into a host cell population, for example, a host cell population in a lagoon, remain within the population for a limited time, and are then removed from the host cell population. In a simple form, a host cell flow may be a flow through a tube, or a channel, for example, at a controlled rate. In other embodiments, a flow of host cells is directed through a lagoon that holds a volume of cell culture media and comprises an inflow and an outflow. The introduction of fresh host cells may be continuous or intermittent and removal may be passive, e.g., by overflow, or active, e.g., by active siphoning or pumping. Removal further may be random, for example, if a stirred suspension culture of host cells is provided, removed liquid culture media will contain freshly introduced host cells as well as cells that have been a member of the host cell population within the lagoon for some time. Even though, in theory, a cell could escape removal from the lagoon indefinitely, the average host cell will remain only for a limited period of time within the lagoon, which is determined mainly by the flow rate of the culture media (and suspended cells) through the lagoon.

Since the viral vectors replicate in a flow of host cells, in which fresh, uninfected host cells are provided while infected cells are removed, multiple consecutive viral life cycles can occur without investigator interaction, which allows for the accumulation of multiple advantageous mutations in a single evolution experiment.

The term “phage-assisted continuous evolution (PACE),” as used herein, refers to continuous evolution that employs phage as viral vectors.

Viral Vectors

The term “viral vector,” as used herein, refers to a nucleic acid comprising a viral genome that, when introduced into a suitable host cell, can be replicated and packaged into viral particles able to transfer the viral genome into another host cell. The term viral vector extends to vectors comprising truncated or partial viral genomes. For example, in some embodiments, a viral vector is provided that lacks a gene encoding a protein essential for the generation of infectious viral particles. In suitable host cells, for example, host cells comprising the lacking gene under the control of a conditional promoter, however, such truncated viral vectors can replicate and generate viral particles able to transfer the truncated viral genome into another host cell. In some embodiments, the viral vector is a phage, for example, a filamentous phage (e.g., an M13 phage). In some embodiments, a viral vector, for example, a phage vector, is provided that comprises a gene of interest to be evolved.

The term “nucleic acid,” as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

The term “protein,” as used herein refers to a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain; see, for example, http://www.cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displays structures of non-natural amino acids that have been successfully incorporated into functional ion channels) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be just a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, or synthetic, or any combination of these.

The term “gene of interest,” as used herein, refers to a nucleic acid construct comprising a nucleotide sequence encoding a gene product of interest, for example, a gene product to be evolved in a continuous evolution process as provided herein. The term includes any variations of a gene of interest that are the result of a continuous evolution process according to methods provided herein. For example, in some embodiments, a gene of interest is a nucleic acid construct comprising a nucleotide sequence encoding a protein to be evolved, cloned into a viral vector, for example, a phage genome, so that the expression of the encoding sequence is under the control of one or more promoters in the viral genome. In other embodiments, a gene of interest is a nucleic acid construct comprising a nucleotide sequence encoding a protein to be evolved and a promoter operably linked to the encoding sequence. When cloned into a viral vector, for example, a phage genome, the expression of the encoding sequence of such genes of interest is under the control of the heterologous promoter and, in some embodiments, may also be influenced by one or more promoters comprised in the viral genome.

The term “function of a gene of interest,” as interchangeably used with the term “activity of a gene of interest,” refers to a function or activity of a gene product, for example, a nucleic acid, or a protein, encoded by the gene of interest. For example, a function of a gene of interest may be an enzymatic activity (e.g., an enzymatic activity resulting in the generation of a reaction product, phosphorylation activity, phosphatase activity, etc.), an ability to activate transcription (e.g., transcriptional activation activity targeted to a specific promoter sequence), a bond-forming activity, (e.g., an enzymatic activity resulting in the formation of a covalent bond), or a binding activity (e.g., a protein, DNA, or RNA binding activity).

The term “promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule “inducer” for activity. Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the instant invention, which is not limited in this respect.

The term “viral particle,” as used herein, refers to a viral genome, for example, a DNA or RNA genome, that is associated with a coat of a viral protein or proteins, and, in some cases, with an envelope of lipids. For example, a phage particle comprises a phage genome packaged into a protein encoded by the wild type phage genome.

The term “infectious viral particle,” as used herein, refers to a viral particle able to transport the viral genome it comprises into a suitable host cell. Not all viral particles are able to transfer the viral genome to a suitable host cell. Particles unable to accomplish this are referred to as a non-infectious viral particles. In some embodiments, a viral particle comprises a plurality of different coat proteins, wherein one or some of the coat proteins can be omitted without compromising the structure of the viral particle. In some embodiments, a viral particle is provided in which at least one coat protein cannot be omitted without the loss of infectivity. If a viral particle lacks a protein that confers infectivity, the viral particle is not infectious. For example, an M13 phage particle that comprises a phage genome packaged in a coat of phage proteins (e.g., pVIII) but lacks pIII (protein III) is a non-infectious M13 phage particle because pIII is essential for the infectious properties of M13 phage particles.

The term “viral life cycle,” as used herein, refers to the viral reproduction cycle comprising insertion of the viral genome into a host cell, replication of the viral genome in the host cell, and packaging of a replication product of the viral genome into a viral particle by the host cell.

In some embodiments, the viral vector provided is a phage. The term “phage,” as used herein interchangeably with the term “bacteriophage,” refers to a virus that infects bacterial cells. Typically, phages consist of an outer protein capsid enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA, in either linear or circular form. Phages and phage vectors are well known to those of skill in the art and non-limiting examples of phages that are useful for carrying out the methods provided herein are λ (Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174, N4, Φ6, and Φ29. In certain embodiments, the phage utilized in the present invention is M13. Additional suitable phages and host cells will be apparent to those of skill in the art and the invention is not limited in this aspect. For an exemplary description of additional suitable phages and host cells, see Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1^stedition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology) Humana Press; 1^stedition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1^stedition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable phages and host cells as well as methods and protocols for isolation, culture, and manipulation of such phages).

In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an M13 phage. M13 phages are well known to those in the art and the biology of M13 phages has extensively been studied. A schematic representation of the wild-type M13 genome is provided in FIG. 16. Wild type M13 phage particles comprise a circular, single-stranded genome of approximately 6.4 kb. The wilt-type genome includes ten genes, gI-gX, which, in turn, encode the ten M13 proteins, pI-pX, respectively. gVIII encodes pVIII, also often referred to as the major structural protein of the phage particles, while gIII encodes pIII, also referred to as the minor coat protein, which is required for infectivity of M13 phage particles.

The M13 life cycle includes attachment of the phage to the sex pilus of a suitable bacterial host cell via the pIII protein and insertion of the phage genome into the host cell. The circular, single-stranded phage genome is then converted to a circular, double-stranded DNA, also termed the replicative form (RF), from which phage gene transcription is initiated. The wild type M13 genome comprises nine promoters and two transcriptional terminators as well as an origin of replication. This series of promoters provides a gradient of transcription such that the genes nearest the two transcriptional terminators (gVIII and IV) are transcribed at the highest levels. In wild-type M13 phage, transcription of all 10 genes proceeds in same direction. One of the phage-encode proteins, pII, initiates the generation of linear, single-stranded phage genomes in the host cells, which are subsequently circularized, and bound and stabilized by pV. The circularized, single-stranded M13 genomes are then bound by pVIII, while pV is stripped off the genome, which initiates the packaging process. At the end of the packaging process, multiple copies of pIII are attached to wild-type M13 particles, thus generating infectious phage ready to infect another host cell and concluding the life cycle.

The M13 phage genome can be manipulated, for example, by deleting one or more of the wild type genes, and/or inserting a heterologous nucleic acid construct into the genome. M13 does not have stringent genome size restrictions, and insertions of up to 42 kb have been reported. This allows M13 phage vectors to be used in continuous evolution experiments to evolve genes of interest without imposing a limitation on the length of the gene to be involved.

The M13 phage has been well characterized and the genomic sequence of M13 has been reported. Representative M13 genomic sequences can be retrieved from public databases and an exemplary sequence is provided in entry V00604 of the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm,nih.gov):

The term “selection phage,” as used herein interchangeably with the term “selection plasmid,” refers to a modified phage that comprises a gene of interest to be evolved and lacks a full-length gene encoding a protein required for the generation of infective phage particles. For example, some M13 selection phage provided herein comprise a nucleic acid sequence encoding a protein to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a phage gene encoding a protein required for the generation of infective phage particles, e.g., gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, or gX, or any combination thereof. For example, some M13 selection phage provided herein comprise a nucleic acid sequence encoding a protein to be evolved, e.g., under the control of an M13 promoter, and lack all or part of a gene encoding a protein required for the generation of infective phage particles, e.g., the gIII gene encoding the pIII protein. An exemplary, non-limiting selection plasmid sequence, SP-MCS, comprising a multiple cloning site, is provided below:

The term “helper phage,” as used herein interchangeable with the terms “helper phagemid” and “helper plasmid,” refers to a nucleic acid construct comprising a phage gene required for the phage life cycle, or a plurality of such genes, but lacking a structural element required for genome packaging into a phage particle. For example, a helper phage may provide a wild-type phage genome lacking a phage origin of replication. In some embodiments, a helper phage is provided that comprises a gene required for the generation of phage particles, but lacks a gene required for the generation of infectious particles, for example, a full-length pIII gene. In some embodiments, the helper phage provides only some, but not all, genes required for the generation of phage particles. Helper phages are useful to allow modified phages that lack a gene required for the generation of phage particles to complete the phage life cycle in a host cell. Typically, a helper phage will comprise the genes required for the generation of phage particles that are lacking in the phage genome, thus complementing the phage genome. In the continuous evolution context, the helper phage typically complements the selection phage, but both lack a phage gene required for the production of infectious phage particles.

The term “replication product,” as used herein; refers to a nucleic acid that is the result of viral genome replication by a host cell. This includes any viral genomes synthesized by the host cell from a viral genome inserted into the host cell. The term includes non-mutated as well as mutated replication products.

Accessory Plasmids and Helper Constructs

The term “accessory plasmid,” as used herein, refers to a plasmid comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter. In the context of continuous evolution described herein, the conditional promoter of the accessory plasmid is typically activated by a function of the gene of interest to be evolved. Accordingly, the accessory plasmid serves the function of conveying a competitive advantage to those viral vectors in a given population of viral vectors that carry a gene of interest able to activate the conditional promoter. Only viral vectors carrying an “activating” gene of interest will be able to induce expression of the gene required to generate infectious viral particles in the host cell, and, thus, allow for packaging and propagation of the viral genome in the flow of host cells. Vectors carrying non-activating versions of the gene of interest, on the other hand, will not induce expression of the gene required to generate infectious viral vectors, and, thus, will not be packaged into viral particles that can infect fresh host cells.

In some embodiments, the conditional promoter of the accessory plasmid is a promote the transcriptional activity of which can be regulated over a wide range, for example, over 2, 3, 4, 5, 6, 7, 8, 9, or 10 orders of magnitude by the activating function, for example, function of a protein encoded by the gene of interest. In some embodiments, the level of transcriptional activity of the conditional promoter depends directly on the desired function of the gene of interest. This allows for starting a continuous evolution process with a viral vector population comprising versions of the gene of interest that only show minimal activation of the conditional promoter. In the process of continuous evolution, any mutation in the gene of interest that increases activity of the conditional promoter directly translates into higher expression levels of the gene required for the generation of infectious viral particles, and, thus, into a competitive advantage over other viral vectors carrying minimally active or loss-of-function versions of the gene of interest.

The stringency of selective pressure imposed by the accessory plasmid in a continuous evolution procedure as provided herein can be modulated. In some embodiments, the use of low copy number accessory plasmids results in an elevated stringency of selection for versions of the gene of interest that activate the conditional promoter on the accessory plasmid, while the use of high copy number accessory plasmids results in a lower stringency of selection. The terms “high copy number plasmid” and “low copy number plasmid” are art-recognized and those of skill in the art will be able to ascertain whether a given plasmid is a high or low copy number plasmid. In some embodiments, a low copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 5 to about 100. In some embodiments, a very low copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 1 to about 10. In some embodiments, a very low copy number accessory plasmid is a single-copy per cell plasmid. In some embodiments, a high copy number accessory plasmid is a plasmid exhibiting an average copy number of plasmid per host cell in a host cell population of about 100 to about 5000. The copy number of an accessory plasmid will depend to a large part on the origin of replication employed. Those of skill in the art will be able to determine suitable origins of replication in order to achieve a desired copy number. The following table lists some non-limiting examples of vectors of different copy numbers and with different origins of replication,

It should be understood that the function of the accessory plasmid, namely to provide a gene required for the generation of viral particles under the control of a conditional promoter the activity of which depends on a function of the gene of interest, can be conferred to a host cell in alternative ways. Such alternatives include, but are not limited to, permanent insertion of a gene construct comprising the conditional promoter and the respective gene into the genome of the host cell, or introducing it into the host cell using an different vector, for example, a phagemid, a cosmid, a phage, a virus, or an artificial chromosome. Additional ways to confer accessory plasmid function to host cells will be evident to those of skill in the art, and the invention is not limited in this respect.

The sequences of two exemplary, non-limiting accessory plasmids, AP-MCS-A, and AP-MCS-P, respectively, are provided below:

The term “mutagen,” as used herein, refers to an agent that induces mutations or increases the rate of mutation in a given biological system, for example, a host cell, to a level above the naturally-occurring level of mutation in that system. Some exemplary mutagens useful for continuous evolution procedures are provided elsewhere herein and other useful mutagens will be evident to those of skill in the art. Useful mutagens include, but are not limited to, ionizing radiation, ultraviolet radiation, base analogs, deaminating agents (e.g., nitrous acid), intercalating agents (e.g., ethidium bromide), alkylating agents (e.g., ethylnitrosourea), transposons, bromine, azide salts, psoralen, benzene, 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no. 77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate (phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7), glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2), N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CAS no. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methane sulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CAS No. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CAS no. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9), N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no. 320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethyl methanesulfonate (EMS) (CAS no. 62-50-0), N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7), 5-diazouracil (CAS no. 2435-76-9) and t-butyl hydroperoxide (BHP) (CAS no. 75-91-2). Additional mutagens can be used in continuous evolution procedures as provided herein, and the invention is not limited in this respect.

Ideally, a mutagen is used at a concentration or level of exposure that induces a desired mutation rate in a given host cell or viral vector population, but is not significantly toxic to the host cells used within the average time frame a host cell is exposed to the mutagen or the time a host cell is present in the host cell flow before being replaced by a fresh host cell.

The term “mutagenesis plasmid,” as used herein, refers to a plasmid comprising a gene encoding a gene product that acts as a mutagen. In some embodiments, the gene encodes a DNA polymerase lacking a proofreading capability. In some embodiments, the gene is a gene involved in the bacterial SOS stress response, for example, a UmuC, UmuD′, or RecA gene.

Host Cells

The term “host cell,” as used herein, refers to a cell that can host a viral vector useful for a continuous evolution process as provided herein. A cell can host a viral vector if it supports expression of genes of viral vector, replication of the viral genome, and/or the generation of viral particles. One criterion to determine whether a cell is a suitable host cell for a given viral vector is to determine whether the cell can support the viral life cycle of a wild-type viral genome that the viral vector is derived from. For example, if the viral vector is a modified M13 phage genome, as provided in some embodiments described herein, then a suitable host cell would be any cell that can support the wild-type M13 phage life cycle. Suitable host cells for viral vectors useful in continuous evolution processes are well known to those of skill in the art, and the invention is not limited in this respect.

In some embodiments, modified viral vectors are used in continuous evolution processes as provided herein. In some embodiments, such modified viral vectors lack a gene required for the generation of infectious viral particles. In some such embodiments, a suitable host cell is a cell comprising the gene required for the generation of infectious viral particles, for example, under the control of a constitutive pr a conditional promoter (e.g., in the form of an accessory plasmid, as described herein). In some embodiments, the viral vector used lacks a plurality of viral genes. In some such embodiments, a suitable host cell is a cell that comprises a helper construct providing the viral genes required for the generation of viral particles. A cell is not required to actually support the life cycle of a viral vector used in the methods provided herein. For example, a cell comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter may not support the life cycle of a viral vector that does not comprise a gene of interest able to activate the promoter, but it is still a suitable host cell for such a viral vector. In some embodiments, the viral vector is a phage and the host cell is a bacterial cell. In some embodiments, the host cell is an E. coli cell. Suitable E. coli host strains will be apparent to those of skill in the art, and include, but are not limited to, New England Biolabs (NEB) Turbo, Top10F′, DH12S, ER2738, ER2267, and XL1-Blue MRF′. These strain names are art recognized and the genotype of these strains has been well characterized. It should be understood that the above strains are exemplary only and that the invention is not limited in this respect.

The term “fresh,” as used herein interchangeably with the terms “non-infected” or “uninfected” in the context of host cells, refers to a host cell that has not been infected by a viral vector comprising a gene of interest as used in a continuous evolution process provided herein. A fresh host cell can, however, have been infected by a viral vector unrelated to the vector to be evolved or by a vector of the same or a similar type but not carrying the gene of interest.

In some embodiments, the host cell is a prokaryotic cell, for example, a bacterial cell. In some embodiments, the host cell is an E. coli cell. In some embodiments, the host cell is a eukaryotic cell, for example, a yeast cell, an insect cell, or a mammalian cell. The type of host cell, will, of course, depend on the viral vector employed, and suitable host cell/viral vector combinations will be readily apparent to those of skill in the art.

In some PACE embodiments, for example, in embodiments employing an M13 selection phage, the host cells are E. coli cells expressing the Fertility factor, also commonly referred to as the F factor, sex factor, or F-plasmid. The F-factor is a bacterial DNA sequence that allows a bacterium to produce a sex pilus necessary for conjugation and is essential for the infection of E. coli cells with certain phage, for example, with M13 phage. For example, in some embodiments, the host cells for M13-PACE are of the genotype F′ proA⁺B⁺ Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻

Lagoons, Cellstats, Turbidostats

The term “lagoon,” as used herein, refers to a vessel through which a flow of host cells is directed. When used for a continuous evolution process as provided herein, a lagoon typically holds a population of host cells and a population of viral vectors replicating within the host cell population, wherein the lagoon comprises an outflow through which host cells are removed from the lagoon and an inflow through which fresh host cells are introduced into the lagoon, thus replenishing the host cell population. In some embodiments, the flow of cells through the lagoon is regulated to result in an essentially constant number of host cells within the lagoon. In some embodiments, the flow of cells through the lagoon is regulated to result in an essentially constant number of fresh host cells within the lagoon.

The term “cellstat,” as used herein, refers to a culture vessel comprising host cells, in which the number of cells is substantially constant over time.

The term “turbidostat,” as used herein, refers to a culture vessel comprising host cells in suspension culture, in which the turbidity of the culture medium is substantially essentially constant over time. In some embodiments, the turbidity of a suspension culture, for example, of bacterial cells, is a measure for the cell density in the culture medium. In some embodiments, a turbidostat comprises an inflow of fresh media and an outflow, and a controller that regulates the flow into and/or out of the turbidostat based on the turbidity of the suspension culture in the turbidostat.

Some aspects of this invention provide methods for the continuous evolution of a nucleic acid, for example, of a gene of interest. Some aspects of this invention provide experimental configurations, systems, apparatuses, reagents, and materials for the continuous evolution methods described herein. Vectors, vector systems, and kits for continuous evolution as described herein are also provided.

Continuous Evolution Methods

The continuous evolution methods provided herein allow for a gene of interest in a viral vector to be evolved over multiple generations of viral life cycles in a flow of host cells to acquire a desired function or activity.

Some aspects of this invention provide a method of continuous evolution of a gene of interest, comprising (a) contacting a population of host cells with a population of viral vectors comprising the gene of interest, wherein (1) the host cell is amenable to infection by the viral vector; (2) the host cell expresses viral genes required for the generation of viral particles; (3) the expression of at least one viral gene required for the production of an infectious viral particle is dependent on a function of the gene of interest; and (4) the viral vector allows for expression of the protein in the host cell, and can be replicated and packaged into a viral particle by the host cell. In some embodiments, the method comprises (b) contacting the host cells with a mutagen. In some embodiments, the method further comprises (c) incubating the population of host cells under conditions allowing for viral replication and the production of viral particles, wherein host cells are removed from the host cell population, and fresh, uninfected host cells are introduced into the population of host cells, thus replenishing the population of host cells and creating a flow of host cells. The cells are incubated in all embodiments under conditions allowing for the gene of interest to acquire a mutation. In some embodiments, the method further comprises (d) isolating a mutated version of the viral vector, encoding an evolved gene product (e.g., protein), from the population of host cells.

In some embodiments, a method of phage-assisted continuous evolution is provided comprising (a) contacting a population of bacterial host cells with a population of phages that comprise a gene of interest to be evolved and that are deficient in a gene required for the generation of infectious phage, wherein (1) the phage allows for expression of the gene of interest in the host cells; (2) the host cells are suitable host cells for phage infection, replication, and packaging; and (3) the host cells comprise an expression construct encoding the gene required for the generation of infectious phage, wherein expression of the gene is dependent on a function of a gene product of the gene of interest. In some embodiments the method further comprises (b) incubating the population of host cells under conditions allowing for the mutation of the gene of interest, the production of infectious phage, and the infection of host cells with phage, wherein infected cells are removed from the population of host cells, and wherein the population of host cells is replenished with fresh host cells that have not been infected by the phage. In some embodiments, the method further comprises (c) isolating a mutated phage replication product encoding an evolved protein from the population of host cells.

In some embodiments, the viral vector or the phage is a filamentous phage, for example, an M13 phage, such as an M13 selection phage as described in more detail elsewhere herein. In some such embodiments, the gene required for the production of infectious viral particles is the M13 gene III (gIII).

In some embodiments, the viral vector infects mammalian cells. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a vesicular stomatitis virus (VSV) vector. As a dsRNA virus, VSV has a high mutation rate, and can carry cargo, including a gene of interest, of up to 4.5 kb in length. The generation of infectious VSV particles requires the envelope protein VSV-G, a viral glycoprotein that mediates phosphatidylserine attachment and cell entry. VSV can infect a broad spectrum of host cells, including mammalian and insect cells. VSV is therefore a highly suitable vector for continuous evolution in human, mouse, or insect host cells. Similarly, other retroviral vectors that can be pseudotyped with VSV-G envelope protein are equally suitable for continuous evolution processes as described herein.

It is known to those of skill in the art that many retroviral vectors, for example, Murine Leukemia Virus vectors, or Lentiviral vectors can efficiently be packaged with VSV-G envelope protein as a substitute for the virus's native envelope protein. In some embodiments, such VSV-G packagable vectors are adapted for use in a continuous evolution system in that the native envelope (env) protein (e.g., VSV-G in VSVS vectors, or env in MLV vectors) is deleted from the viral genome, and a gene of interest is inserted into the viral genome under the control of a promoter that is active in the desired host cells. The host cells, in turn, express the VSV-G protein, another env protein suitable for vector pseudotyping, or the viral vector's native env protein, under the control of a promoter the activity of which is dependent on an activity of a product encoded by the gene of interest, so that a viral vector with a mutation leading to an increased activity of the gene of interest will be packaged with higher efficiency than a vector with baseline or a loss-of-function mutation.

In some embodiments, mammalian host cells are subjected to infection by a continuously evolving population of viral vectors, for example, VSV vectors comprising a gene of interest and lacking the VSV-G encoding gene, wherein the host cells comprise a gene encoding the VSV-G protein under the control of a conditional promoter. Such retrovirus-bases system could be a two-vector system (the viral vector and an expression construct comprising a gene encoding the envelope protein), or, alternatively, a helper virus can be employed, for example, a VSV helper virus. A helper virus typically comprises a truncated viral genome deficient of structural elements required to package the genome into viral particles, but including viral genes encoding proteins required for viral genome processing in the host cell, and for the generation of viral particles. In such embodiments, the viral vector-based system could be a three-vector system (the viral vector, the expression construct comprising the envelope protein driven by a conditional promoter, and the helper virus comprising viral functions required for viral genome propagation but not the envelope protein). In some embodiments, expression of the five genes of the VSV genome from a helper virus or expression construct in the host cells, allows for production of infectious viral particles carrying a gene of interest, indicating that unbalanced gene expression permits viral replication at a reduced rate, suggesting that reduced expression of VSV-G would indeed serve as a limiting step in efficient viral production.

One advantage of using a helper virus is that the viral vector can be deficient in genes encoding proteins or other functions provided by the helper virus, and can, accordingly, carry a longer gene of interest. In some embodiments, the helper virus does not express an envelope protein, because expression of a viral envelope protein is known to reduce the infectability of host cells by some viral vectors via receptor interference. Viral vectors, for example retroviral vectors, suitable for continuous evolution processes, their respective envelope proteins, and helper viruses for such vectors, are well known to those of skill in the art. For an overview of some exemplary viral genomes, helper viruses, host cells, and envelope proteins suitable for continuous evolution procedures as described herein, see Coffin et al., Retroviruses, CSHL Press 1997, ISBN0-87969-571-4, incorporated herein in its entirety.

In some embodiments, the incubating of the host cells is for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles. In certain embodiments, the viral vector is an M13 phage, and the length of a single viral life cycle is about 10-20 minutes.

In some embodiments, the cells are contacted and/or incubated in suspension culture. For example, in some embodiments, bacterial cells are incubated in suspension culture in liquid culture media. Suitable culture media for bacterial suspension culture will be apparent to those of skill in the art, and the invention is not limited in this regard. See, for example, Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); Elizabeth Kutter and Alexander Sulakvelidze: Bacteriophages: Biology and Applications. CRC Press; 1^stedition (December 2004), ISBN: 0849313368; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions (Methods in Molecular Biology) Humana Press; 1^stedition (December, 2008), ISBN: 1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methods in Molecular Biology) Humana Press; 1^stedition (December 2008), ISBN: 1603275649; all of which are incorporated herein in their entirety by reference for disclosure of suitable culture media for bacterial host cell culture). Suspension culture typically requires the culture media to be agitated, either continuously or intermittently. This is achieved, in some embodiments, by agitating or stirring the vessel comprising the host cell population. In some embodiments, the outflow of host cells and the inflow of fresh host cells is sufficient to maintain the host cells in suspension. This in particular, if the flow rate of cells into and/or out of the lagoon is high.

In some embodiments, a viral vector/host cell combination is chosen in which the life cycle of the viral vector is significantly shorter than the average time between cell divisions of the host cell. Average cell division times and viral vector life cycle times are well known in the art for many cell types and vectors, allowing those of skill in the art to ascertain such host cell/vector combinations. In certain embodiments, host cells are being removed from the population of host cells contacted with the viral vector at a rate that results in the average time of a host cell remaining in the host cell population before being removed to be shorter than the average time between cell divisions of the host cells, but to be longer than the average life cycle of the viral vector employed. The result of this is that the host cells, on average, do not have sufficient time to proliferate during their time in the host cell population while the viral vectors do have sufficient time to infect a host cell, replicate in the host cell, and generate new viral particles during the time a host cell remains in the cell population. This assures that the only replicating nucleic acid in the host cell population is the viral vector, and that the host cell genome, the accessory plasmid, or any other nucleic acid constructs cannot acquire mutations allowing for escape from the selective pressure imposed.

For example, in some embodiments, the average time a host cell remains in the host cell population is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes.

In some embodiments, the average time a host cell remains in the host cell population depends on how fast the host cells divide and how long infection (or conjugation) requires. In general, the flow rate should be faster than the average time required for cell division, but slow enough to allow viral (or conjugative) propagation. The former will vary, for example, with the media type, and can be delayed by adding cell division inhibitor antibiotics (FtsZ inhibitors in E. coli, etc.). Since the limiting step in continuous evolution is production of the protein required for gene transfer from cell to cell, the flow rate at which the vector washes out will depend on the current activity of the gene(s) of interest. In some embodiments, titratable production of the protein required for the generation of infectious particles, as described herein, can mitigate this problem. In some embodiments, an indicator of phage infection allows computer-controlled optimization of the flow rate for the current activity level in real-time.

In some embodiments, the host cell population is continuously replenished with fresh, uninfected host cells. In some embodiments, this is accomplished by a steady stream of fresh host cells into the population of host cells. In other embodiments, however, the inflow of fresh host cells into the lagoon is semi-continuous or intermittent (e.g., batch-fed). In some embodiments, the rate of fresh host cell inflow into the cell population is such that the rate of removal of cells from the host cell population is compensated. In some embodiments, the result of this cell flow compensation is that the number of cells in the cell population is substantially constant over the time of the continuous evolution procedure. In some embodiments, the portion of fresh, uninfected cells in the cell population is substantially constant over the time of the continuous evolution procedure. For example, in some embodiments, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, or about 90% of the cells in the host cell population are not infected by virus. In general, the faster the flow rate of host cells is, the smaller the portion of cells in the host cell population that are infected will be. However, faster flow rates allow for more transfer cycles, e.g., viral life cycles, and, thus, for more generations of evolved vectors in a given period of time, while slower flow rates result in a larger portion of infected host cells in the host cell population and therefore a larger library size at the cost of slower evolution. In some embodiments, the range of effective flow rates is invariably bounded by the cell division time on the slow end and vector washout on the high end In some embodiments, the viral load, for example, as measured in infectious viral particles per volume of cell culture media is substantially constant over the time of the continuous evolution procedure.

In some embodiments, the fresh host cells comprise the accessory plasmid required for selection of viral vectors, for example, the accessory plasmid comprising the gene required for the generation of infectious phage particles that is lacking from the phages being evolved. In some embodiments, the host cells are generated by contacting an uninfected host cell with the relevant vectors, for example, the accessory plasmid and, optionally, a mutagenesis plasmid, and growing an amount of host cells sufficient for the replenishment of the host cell population in a continuous evolution experiment. Methods for the introduction of plasmids and other gene constructs into host cells are well known to those of skill in the art and the invention is not limited in this respect. For bacterial host cells, such methods include, but are not limited to electroporation and heat-shock of competent cells. In some embodiments, the accessory plasmid comprises a selection marker, for example, an antibiotic resistance marker, and the fresh host cells are grown in the presence of the respective antibiotic to ensure the presence of the plasmid in the host cells. Where multiple plasmids are present, different markers are typically used. Such selection markers and their use in cell culture are known to those of skill in the art, and the invention is not limited in this respect.

In some embodiments, the host cell population in a continuous evolution experiment is replenished with fresh host cells growing in a parallel, continuous culture. In some embodiments, the cell density of the host cells in the host cell population contacted with the viral vector and the density of the fresh host cell population is substantially the same.

Typically, the cells being removed from the cell population contacted with the viral vector comprise cells that are infected with the viral vector and uninfected cells. In some embodiments, cells are being removed from the cell populations continuously, for example, by effecting a continuous outflow of the cells from the population. In other embodiments, cells are removed semi-continuously or intermittently from the population. In some embodiments, the replenishment of fresh cells will match the mode of removal of cells from the cell population, for example, if cells are continuously removed, fresh cells will be continuously introduced. However, in some embodiments, the modes of replenishment and removal may be mismatched, for example, a cell population may be continuously replenished with fresh cells, and cells may be removed semi-continuously or in batches.

In some embodiments, the rate of fresh host cell replenishment and/or the rate of host cell removal is adjusted based on quantifying the host cells in the cell population. For example, in some embodiments, the turbidity of culture media comprising the host cell population is monitored and, if the turbidity falls below a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect an increase in the number of host cells in the population, as manifested by increased cell culture turbidity. In other embodiments, if the turbidity rises above a threshold level, the ratio of host cell inflow to host cell outflow is adjusted to effect a decrease in the number of host cells in the population, as manifested by decreased cell culture turbidity. Maintaining the density of host cells in the host cell population within a specific density range ensures that enough host cells are available as hosts for the evolving viral vector population, and avoids the depletion of nutrients at the cost of viral packaging and the accumulation of cell-originated toxins from overcrowding the culture.

In some embodiments, the cell density in the host cell population and/or the fresh host cell density in the inflow is about 10²cells/ml to about 10¹²cells/ml. In some embodiments, the host cell density is about 10²cells/ml, about 10³cells/ml, about 10⁴cells/ml, about 10⁵cells/ml, about 5.10⁵cells/ml, about 10⁶cells/ml, about 5.10⁶cells/ml, about 10⁷cells/ml, about 5.10⁷cells/ml, about 10⁸cells/ml, about 5.10⁸cells/ml, about 10⁹cells/ml, about 5.10⁹cells/ml, about 10¹⁰cells/ml, or about 5.10¹⁰cells/ml. In some embodiments, the host cell density is more than about 10¹⁰cells/ml.

In some embodiments, the host cell population is contacted with a mutagen. In some embodiments, the cell population contacted with the viral vector (e.g., the phage), is continuously exposed to the mutagen at a concentration that allows for an increased mutation rate of the gene of interest, but is not significantly toxic for the host cells during their exposure to the mutagen while in the host cell population. In other embodiments, the host cell population is contacted with the mutagen intermittently, creating phases of increased mutagenesis, and accordingly, of increased viral vector diversification. For example, in some embodiments, the host cells are exposed to a concentration of mutagen sufficient to generate an increased rate of mutagenesis in the gene of interest for about 10%, about 20%, about 50%, or about 75% of the time.

In some embodiments, the host cells comprise a mutagenesis expression construct, for example, in the case of bacterial host cells, a mutagenesis plasmid. In some embodiments, the mutagenesis plasmid comprises a gene expression cassette encoding a mutagenesis-promoting gene product, for example, a proofreading-impaired DNA polymerase. In other embodiments, the mutagenesis plasmid, including a gene involved in the SOS stress response, (e.g., UmuC, UmuD′, and/or RecA). In some embodiments, the mutagenesis-promoting gene is under the control of an inducible promoter. Suitable inducible promoters are well known to those of skill in the art and include, for example, arabinose-inducible promoters, tetracycline or doxycyclin-inducible promoters, and tamoxifen-inducible promoters. In some embodiments, the host cell population is contacted with an inducer of the inducible promoter in an amount sufficient to effect an increased rate of mutagenesis. For example, in some embodiments, a bacterial host cell population is provided in which the host cells comprise a mutagenesis plasmid in which a dnaQ926, UmuC, UmuD′, and RecA expression cassette is controlled by an arabinose-inducible promoter. In some such embodiments, the population of host cells is contacted with the inducer, for example, arabinose in an amount sufficient to induce an increased rate of mutation.

The use of an inducible mutagenesis plasmid allows one to generate a population of fresh, uninfected host cells in the absence of the inducer, thus avoiding an increased rate of mutation in the fresh host cells before they are introduced into the population of cells contacted with the viral vector. Once introduced into this population, however, these cells can then be induced to support an increased rate of mutation, which is particularly useful in some embodiments of continuous evolution. For example, in some embodiments, the host cell comprise a mutagenesis plasmid as described herein, comprising an arabinose-inducible promoter driving expression of dnaQ926, UmuC, UmuD′, and RecA730 from a pBAD promoter (see, e.g., Khlebnikov A, Skaug T, Keasling J D. Modulation of gene expression from the arabinose-inducible araBAD promoter. J Ind Microbiol Biotechnol. 2002 July; 29(1):34-7; incorporated herein by reference for disclosure of a pBAD promoter). In some embodiments, the fresh host cells are not exposed to arabinose, which activates expression of the above identified genes and, thus, increases the rate of mutations in the arabinose-exposed cells, until the host cells reach the lagoon in which the population of selection phage replicates. Accordingly, in some embodiments, the mutation rate in the host cells is normal until they become part of the host cell population in the lagoon, where they are exposed to the inducer (e.g., arabinose) and, thus, to increased mutagenesis. In some embodiments, a method of continuous evolution is provided that includes a phase of diversifying the population of viral vectors by mutagenesis, in which the cells are incubated under conditions suitable for mutagenesis of the viral vector in the absence of stringent selection for the mutated replication product of the viral vector encoding the evolved protein. This is particularly useful in embodiments in which a desired function to be evolved is not merely an increase in an already present function, for example, an increase in the transcriptional activation rate of a transcription factor, but the acquisition of a function not present in the gene of interest at the outset of the evolution procedure. A step of diversifying the pool of mutated versions of the gene of interest within the population of viral vectors, for example, of phage, allows for an increase in the chance to find a mutation that conveys the desired function.

In some embodiments, diversifying the viral vector population is achieved by providing a flow of host cells that does not select for gain-of-function mutations in the gene of interest for replication, mutagenesis, and propagation of the population of viral vectors. In some embodiments, the host cells are host cells that express all genes required for the generation of infectious viral particles, for example, bacterial cells that express a complete helper phage, and, thus, do not impose selective pressure on the gene of interest. In other embodiments, the host cells comprise an accessory plasmid comprising a conditional promoter with a baseline activity sufficient to support viral vector propagation even in the absence of significant gain-of-function mutations of the gene of interest. This can be achieved by using a “leaky” conditional promoter, by using a high-copy number accessory plasmid, thus amplifying baseline leakiness, and/or by using a conditional promoter on which the initial version of the gene of interest effects a low level of activity while a desired gain-of-function mutation effects a significantly higher activity.

For example, as described in more detail in the Example section, in some embodiments, a population of host cells comprising a high-copy accessory plasmid with a gene required for the generation of infectious phage particles is contacted with a selection phage comprising a gene of interest, wherein the accessory plasmid comprises a conditional promoter driving expression of the gene required for the generation from a conditional promoter, the activity of which is dependent on the activity of a gene product encoded by the gene of interest. In some such embodiments, a low stringency selection phase can be achieved by designing the conditional promoter in a way that the initial gene of interest exhibits some activity on that promoter. For example, if a transcriptional activator, such as a T7RNAP or a transcription factor is to be evolved to recognize a non-native target DNA sequence (e.g., a T3RNAP promoter sequence, on which T7RNAP has no activity), a low-stringency accessory plasmid can be designed to comprise a conditional promoter in which the target sequence comprises a desired characteristic, but also retains a feature of the native recognition sequence that allows the transcriptional activator to recognize the target sequence, albeit with less efficiency than its native target sequence. Initial exposure to such a low-stringency accessory plasmid comprising a hybrid target sequence (e.g., a T7/T3 hybrid promoter, with some features of the ultimately desired target sequence and some of the native target sequence) allows the population of phage vectors to diversify by acquiring a plurality of mutations that are not immediately selected against based on the permissive character of the accessory plasmid. Such a diversified population of phage vectors can then be exposed to a stringent selection accessory plasmid, for example, a plasmid comprising in its conditional promoter the ultimately desired target sequence that does not retain a feature of the native target sequence, thus generating a strong negative selective pressure against phage vectors that have not acquired a mutation allowing for recognition of the desired target sequence.

In some embodiments, an initial host cell population contacted with a population of evolving viral vectors is replenished with fresh host cells that are different from the host cells in the initial population. For example, in some embodiments, the initial host cell population is made of host cells comprising a low-stringency accessory plasmid, or no such plasmid at all, or are permissible for viral infection and propagation. In some embodiments, after diversifying the population of viral vectors in the low-stringency or no-selection host cell population, fresh host cells are introduced into the host cell population that impose a more stringent selective pressure for the desired function of the gene of interest. For example, in some embodiments, the secondary fresh host cells are not permissible for viral replication and propagation anymore. In some embodiments, the stringently selective host cells comprise an accessory plasmid in which the conditional promoter exhibits none or only minimal baseline activity, and/or which is only present in low or very low copy numbers in the host cells.

Such methods involving host cells of varying selective stringency allow for harnessing the power of continuous evolution methods as provided herein for the evolution of functions that are completely absent in the initial version of the gene of interest, for example, for the evolution of a transcription factor recognizing a foreign target sequence that a native transcription factor, used as the initial gene of interest, does not recognize at all. Or, for another example, the recognition of a desired target sequence by a DNA-binding protein, a recombinase, a nuclease, a zinc-finger protein, or an RNA-polymerase, that does not bind to or does not exhibit any activity directed towards the desired target sequence.

In some embodiments, negative selection is applied during a continuous evolution method as described herein, by penalizing undesired activities. In some embodiments, this is achieved by causing the undesired activity to interfere with pIII production. For example, expression of an antisense RNA complementary to the gIII RBS and/or start codon is one way of applying negative selection, while expressing a protease (e.g., TEV) and engineering the protease recognition sites into pIII is another.

In some embodiments, negative selection is applied during a continuous evolution method as described herein, by penalizing the undesired activities of evolved products. This is useful, for example, if the desired evolved product is an enzyme with high specificity, for example, a transcription factor or protease with altered, but not broadened, specificity. In some embodiments, negative selection of an undesired activity is achieved by causing the undesired activity to interfere with pIII production, thus inhibiting the propagation of phage genomes encoding gene products with an undesired activity. In some embodiments, expression of a dominant-negative version of pIII or expression of an antisense RNA complementary to the gIII RBS and/or gIII start codon is linked to the presence of an undesired activity. In some embodiments, a nuclease or protease cleavage site, the recognition or cleavage of which is undesired, is inserted into a pIII transcript sequence or a pIII amino acid sequence, respectively. In some embodiments, a transcriptional or translational repressor is used that represses expression of a dominant negative variant of pIII and comprises a protease cleavage site the recognition or cleaveage of which is undesired.

In some embodiments, counter-selection against activity on non-target substrates is achieved by linking undesired evolved product activities to the inhibition of phage propagation. For example, in some embodiments, in which a transcription factor is evolved to recognize a specific target sequence, but not an undesired off-target sequence, a negative selection cassette is employed, comprising a nucleic acid sequence encoding a dominant-negative version of pIII (pIII-neg) under the control of a promoter comprising the off-target sequence. If an evolution product recognizes the off-target sequence, the resulting phage particles will incorporate pIII-neg, which results in an inhibition of phage infective potency and phage propagation, thus constituting a selective disadvantage for any phage genomes encoding an evolution product exhibiting the undesired, off-target activity, as compared to evolved products not exhibiting such an activity. In some embodiments, a dual selection strategy is applied during a continuous evolution experiment, in which both positive selection and negative selection constructs are present in the host cells. In some such embodiments, the positive and negative selection constructs are situated on the same plasmid, also referred to as a dual selection accessory plasmid.

For example, in some embodiments, a dual selection accessory plasmid is employed comprising a positive selection cassette, comprising a pIII-encoding sequence under the control of a promoter comprising a target nucleic acid sequence, and a negative selection cassette, comprising a pIII-neg encoding cassette under the control of a promoter comprising an off-target nucleic acid sequence. One advantage of using a simultaneous dual selection strategy is that the selection stringency can be fine-tuned based on the activity or expression level of the negative selection construct as compared to the positive selection construct. Another advantage of a dual selection strategy is the selection is not dependent on the presence or the absence of a desired or an undesired activity, but on the ratio of desired and undesired activities, and, thus, the resulting ratio of pIII and pIII-neg that is incorporated into the respective phage particle.

Some aspects of this invention provide or utilize a dominant negative variant of pIII (pIII-neg). These aspects are based on the surprising discovery that a pIII variant that comprises the two N-terminal domains of pIII and a truncated, termination-incompetent C-terminal domain is not only inactive but is a dominant-negative variant of pIII. A pIII variant comprising the two N-terminal domains of pIII and a truncated, termination-incompetent C-terminal domain was described in Bennett, N.J.; Rakonjac, J., Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII. Journal of Molecular Biology 2006, 356 (2), 266-73; the entire contents of which are incorporated herein by reference. However, the dominant negative property of such pIII variants has not been previously described. Some aspects of this invention are based on the surprising discovery that a pIII-neg variant as provided herein is efficiently incorporated into phage particles, but it does not catalyze the unlocking of the particle for entry during infection, rendering the respective phage noninfectious even if wild type pIII is present in the same phage particle. Accordingly, such pIII-neg variants are useful for devising a negative selection strategy in the context of PACE, for example, by providing an expression construct comprising a nucleic acid sequence encoding a pIII-neg variant under the control of a promoter comprising a recognition motif, the recognition of which is undesired. In other embodiments, pIII-neg is used in a positive selection strategy, for example, by providing an expression construct in which a pIII-neg encoding sequence is controlled by a promoter comprising a nuclease target site or a repressor recognition site, the recognition of either one is desired.

Positive and negative selection strategies can further be designed to link non-DNA directed activities to phage propagation efficiency. For example, protease activity towards a desired target protease cleavage site can be linked to pIII expression by devising a repressor of gene expression that can be inactivated by a protease recognizing the target site. In some embodiments, pIII expression is driven by a promoter comprising a binding site for such a repressor. Suitable transcriptional repressors are known to those in the art, and one exemplary repressor is the lambda repressor protein, that efficiently represses the lambda promoter pR and can be modified to include a desired protease cleavage site (see, e.g., Sices, H. J.; Kristie, T. M., A genetic screen for the isolation and characterization of site-specific proteases. Proc Natl Acad Sci USA 1998, 95 (6), 2828-33; and Sices, H. J.; Leusink, M. D.; Pacheco, A.; Kristie, T. M., Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease. AIDS Res Hum Retroviruses 2001, 17 (13), 1249-55, the entire contents of each of which are incorporated herein by reference). The lambda repressor (cI) contains an N-terminal DNA binding domain and a C-terminal dimerization domain. These two domains are connected by a flexible linker. Efficient transcriptional repression requires the dimerization of cI, and, thus, cleavage of the linker connecting dimerization and binding domains results in abolishing the repressor activity of cI.

Some embodiments provide a pIII expression construct that comprises a pR promoter (containing cI binding sites) driving expression of pIII. When expressed together with a modified cI comprising a desired protease cleavage site in the linker sequence connecting dimerization and binding domains, the cI molecules will repress pIII transcription in the absence of the desired protease activity, and this repression will be abolished in the presence of such activity, thus providing a linkage between protease cleavage activity and an increase in pIII expression that is useful for positive PACE protease selection. Some embodiments provide a negative selection strategy against undesired protease activity in PACE evolution products. In some embodiments, the negative selection is conferred by an expression cassette comprising a pIII-neg encoding nucleic acid under the control of a cI-repressed promoter. When co-expressed with a cI repressor protein comprising an undesired protease cleavage site, expression of pIII-neg will occur in cell harboring phage expressing a protease exhibiting protease activity towards the undesired target site, thus negatively selecting against phage encoding such undesired evolved products. A dual selection for protease target specificity can be achieved by co-expressing cI-repressible pIII and pIII-neg encoding expression constructs with orthogonal cI variants recognizing different DNA target sequences, and thus allowing for simultaneous expression without interfering with each other. Orthogonal cI variants in both dimerization specificity and DNA-binding specificity are known to those of skill in the art (see, e.g., Wharton, R. P.; Ptashne, M., Changing the binding specificity of a repressor by redesigning an alphahelix. Nature 1985, 316 (6029), 601-5; and Wharton, R. P.; Ptashne, M., A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact. Nature 1987, 326 (6116), 888-91, the entire contents of each of which are incorporated herein by reference).

Other selection schemes for gene products having a desired activity are well known to those of skill in the art or will be apparent from the instant disclosure. Selection strategies that can be used in continuous evolution processes and methods as provided herein include, but are not limited to, selection strategies useful in two-hybrid screens. For example, the T7 RNAP selection strategy described in more detail elsewhere herein is an example of a promoter recognition selection strategy. Two-hybrid accessory plasmid setups further permit the evolution of protein-protein interactions, and accessory plasmids requiring site-specific recombinase activity for production of the protein required for the generation of infectious viral particles, for example, pIII, allow recombinases to be evolved to recognize any desired target site. A two-hybrid setup or a related one-hybrid setup can further be used to evolve DNA-binding proteins, while a three-hybrid setup can evolve RNA-protein interactions.

Biosynthetic pathways producing small molecules can also be evolved with a promoter or riboswitch (e.g., controlling gene III expression/translation) that is responsive to the presence of the desired small molecule. For example, a promoter that is transcribed only in the presence of butanol could be placed on the accessory plasmid upstream of gene III to optimize a biosynthetic pathway encoding the enzymes for butanol synthesis. A phage vector carrying a gene of interest that has acquired an activity boosting butanol synthesis would have a selective advantage over other phages in an evolving phage population that have not acquired such a gain-of-function. Alternatively, a chemical complementation system, for example, as described in Baker and Cornish, PNAS, 2002, incorporated herein by reference, can be used to evolve individual proteins or enzymes capable of bond formation reactions 0. In other embodiments, a trans-splicing intron designed to splice itself into a particular target sequence can be evolved by expressing only the latter half of gene III from the accessory plasmid, preceded by the target sequence, and placing the other half (fused to the trans-splicing intron) on the selection phage. Successful splicing would reconstitute full-length pIII-encoding mRNA. Protease specificity and activity can be evolved by expressing pIII fused to a large protein from the accessory plasmid, separated by a linker containing the desired protease recognition site. Cleavage of the linker by active protease encoded by the selection phage would result in infectious pIII, while uncleaved pIII would be unable to bind due to the blocking protein. Further, As described, for example, by Malmborg and Borrebaeck 1997, a target antigen can be fused to the F pilus of a bacteria, blocking wild-type pIII from binding. Phage displaying antibodies specific to the antigen could bind and infect, yielding enrichments of >1000-fold in phage display. In some embodiments, this system can be adapted for continuous evolution, in that the accessory plasmid is designed to produce wild-type pIII to contact the tolA receptor and perform the actual infection (as the antibody-pIII fusion binds well but infects with low efficiency), while the selection phage encodes the pIII-antibody fusion protein. Progeny phage containing both types of pIII tightly adsorb to the F pilus through the antibody-antigen interaction, with the wild-type pIII contacting tolA and mediating high-efficiency infection. To allow propagation when the initial antibody-antigen interaction is weak, a mixture of host cells could flow into the lagoon: a small fraction expressing wild-type pili and serving as a reservoir of infected cells capable of propagating any selection phage regardless of activity, while the majority of cells requires a successful interaction, serving as the “reward” for any mutants that improve their binding affinity. This last system, in some embodiments, can evolve new antibodies that are effective against a target pathogen faster than the pathogen itself can evolve, since the evolution rates of PACE and other systems described herein are higher than those of human-specific pathogens, for example, those of human viruses.

Methods and strategies to design conditional promoters suitable for carrying out the selections strategies described herein are well known to those of skill in the art. Some exemplary design strategies are summarized in FIG. 3B. For an overview over exemplary suitable selection strategies and methods for designing conditional promoters driving the expression of a gene required for cell-cell gene transfer, e.g. gIII, see Vidal and Legrain, Yeast n-hybrid review, Nucleic Acid Research 27, 919 (1999), incorporated herein in its entirety.

Apparatus for Continued Evolution

The invention also provides apparatuses for continuous evolution of a nucleic acid. The core element of such an apparatus is a lagoon allowing for the generation of a flow of host cells in which a population of viral vectors can replicate and propagate. In some embodiments, the lagoon comprises a cell culture vessel comprising an actively replicating population of viral vectors, for example, phage vectors comprising a gene of interest, and a population of host cells, for example, bacterial host cells. In some embodiments, the lagoon comprises an inflow for the introduction of fresh host cells into the lagoon and an outflow for the removal of host cells from the lagoon. In some embodiments, the inflow is connected to a turbidostat comprising a culture of fresh host cells. In some embodiments, the outflow is connected to a waste vessel, or a sink. In some embodiments, the lagoon further comprises an inflow for the introduction of a mutagen into the lagoon. In some embodiments that inflow is connected to a vessel holding a solution of the mutagen. In some embodiments, the lagoon comprises an inflow for the introduction of an inducer of gene expression into the lagoon, for example, of an inducer activating an inducible promoter within the host cells that drives expression of a gene promoting mutagenesis (e.g., as part of a mutagenesis plasmid), as described in more detail elsewhere herein. In some embodiments, that inflow is connected to a vessel comprising a solution of the inducer, for example, a solution of arabinose.

In some embodiments, the lagoon comprises a population of viral vectors. In some embodiments, the lagoon comprises a population of viral vectors. In some embodiments, the viral vectors are phage, for example, M13 phages deficient in a gene required for the generation of infectious viral particles as described herein. In some such embodiments, the host cells are prokaryotic cells amenable to phage infection, replication, and propagation of phage, for example, host cells comprising an accessory plasmid comprising a gene required for the generation of infectious viral particles under the control of a conditional promoter as described herein.

In some embodiments, the lagoon comprises a controller for regulation of the inflow and outflow rates of the host cells, the inflow of the mutagen, and/or the inflow of the inducer. In some embodiments, a visual indicator of phage presence, for example, a fluorescent marker, is tracked and used to govern the flow rate, keeping the total infected population constant. In some embodiments, the visual marker is a fluorescent protein encoded by the phage genome, or an enzyme encoded by the phage genome that, once expressed in the host cells, results in a visually detectable change in the host cells. In some embodiments, the visual tracking of infected cells is used to adjust a flow rate to keep the system flowing as fast as possible without risk of vector washout.

In some embodiments, the expression of the gene required for the generation of infectious particles is titratable. In some embodiments, this is accomplished with an accessory plasmid producing pIII proportional to the amount of anhydrotetracycline added to the lagoon. Other In some embodiments, such a titrable expression construct can be combined with another accessory plasmid as described herein, allowing simultaneous selection for activity and titratable control of pIII. This permits the evolution of activities too weak to otherwise survive in the lagoon, as well as allowing neutral drift to escape local fitness peak traps. In some embodiments, negative selection is applied during a continuous evolution method as described herein, by penalizing undesired activities. In some embodiments, this is achieved by causing the undesired activity to interfere with pIII production. For example, expression of an antisense RNA complementary to the gIII RBS and/or start codon is one way of applying negative selection, while expressing a protease (e.g., TEV) and engineering the protease recognition sites into pIII is another.

In some embodiments, the apparatus comprises a turbidostat. In some embodiments, the turbidostat comprises a cell culture vessel in which the population of fresh host cells is situated, for example, in liquid suspension culture. In some embodiments, the turbidostat comprises an outflow that is connected to an inflow of the lagoon, allowing the introduction of fresh cells from the turbidostat into the lagoon. In some embodiments, the turbidostat comprises an inflow for the introduction of fresh culture media into the turbidostat. In some embodiments, the inflow is connected to a vessel comprising sterile culture media. In some embodiments, the turbidostat further comprises an outflow for the removal of host cells from the turbidostat. In some embodiments, that outflow is connected to a waste vessel or drain.

In some embodiments, the turbidostat comprises a turbidity meter for measuring the turbidity of the culture of fresh host cells in the turbidostat. In some embodiments, the turbidostat comprises a controller that regulated the inflow of sterile liquid media and the outflow into the waste vessel based on the turbidity of the culture liquid in the turbidostat.

In some embodiments, the lagoon and/or the turbidostat comprises a shaker or agitator for constant or intermittent agitation, for example, a shaker, mixer, stirrer, or bubbler, allowing for the population of host cells to be continuously or intermittently agitated and oxygenated.

In some embodiments, the controller regulates the rate of inflow of fresh host cells into the lagoon to be substantially the same (volume/volume) as the rate of outflow from the lagoon. In some embodiments, the rate of inflow of fresh host cells into and/or the rate of outflow of host cells from the lagoon is regulated to be substantially constant over the time of a continuous evolution experiment. In some embodiments, the rate of inflow and/or the rate of outflow is from about 0.1 lagoon volumes per hour to about 25 lagoon volumes per hour. In some embodiments, the rate of inflow and/or the rate of outflow is approximately 0.1 lagoon volumes per hour (lv/h), approximately 0.2 lv/h, approximately 0.25 lv/h, approximately 0.3 lv/h, approximately 0.4 lv/h, approximately 0.5 lv/h, approximately 0.6 lv/h, approximately 0.7 lv/h, approximately 0.75 lv/h, approximately 0.8 lv/h, approximately 0.9 lv/h, approximately 1 lv/h, approximately 2 lv/h, approximately 2.5 lv/h, approximately 3 lv/h, approximately 4 lv/h, approximately 5 lv/h, approximately 7.5 lv/h, approximately 10 lv/h, or more than 10 lv/h.

In some embodiments, the inflow and outflow rates are controlled based on a quantitative assessment of the population of host cells in the lagoon, for example, by measuring the cell number, cell density, wet biomass weight per volume, turbidity, or cell growth rate. In some embodiments, the lagoon inflow and/or outflow rate is controlled to maintain a host cell density of from about 10²cells/ml to about 10¹²cells/ml in the lagoon. In some embodiments, the inflow and/or outflow rate is controlled to maintain a host cell density of about 10²cells/ml, about 10³cells/ml, about 10⁴cells/ml, about 10⁵cells/ml, about 5×10⁵cells/ml, about 10⁶cells/ml, about 5×10⁶cells/ml, about 10⁷cells/ml, about 5×10⁷cells/ml, about 10⁸cells/ml, about 5×10⁸cells/ml, about 10⁹cells/ml, about 5×10⁹cells/ml, about 10¹⁰cells/ml, about 5×10¹⁰cells/ml, or more than 5×10¹⁰cells/ml, in the lagoon. In some embodiments, the density of fresh host cells in the turbidostat and the density of host cells in the lagoon are substantially identical.

In some embodiments, the lagoon inflow and outflow rates are controlled to maintain a substantially constant number of host cells in the lagoon. In some embodiments, the inflow and outflow rates are controlled to maintain a substantially constant frequency of fresh host cells in the lagoon. In some embodiments, the population of host cells is continuously replenished with fresh host cells that are not infected by the phage. In some embodiments, the replenishment is semi-continuous or by batch-feeding fresh cells into the cell population.

In some embodiments, the lagoon volume is from approximately 1 ml to approximately 100 l, for example, the lagoon volume is approximately 1 ml, approximately 10 ml, approximately 50 ml, approximately 100 ml, approximately 200 ml, approximately 250 ml, approximately 500 ml, approximately 750 ml, approximately 1 l, approximately 2 ml, approximately 2.5 l, approximately 3 l, approximately 4 l, approximately 5 l, approximately 10 l, approximately 1 ml-10 ml, approximately 10 ml-50 ml, approximately 50 ml-100, approximately 100 ml-250 ml, approximately 250 ml-500 ml, approximately 500 ml-1 l, approximately 1 l-2 l, approximately 2 l-5 l, approximately 5 l-10 l, approximately 10-50 l, approximately 50-100 l, or more than 100 l.

In some embodiments, the lagoon and/or the turbidostat further comprises a heater and a thermostat controlling the temperature. In some embodiments, the temperature in the lagoon and/or the turbidostat is controlled to be from about 4° C. to about 55° C., preferably from about 25° C. to about 39° C., for example, about 37° C.

In some embodiments, the inflow rate and/or the outflow rate is controlled to allow for the incubation and replenishment of the population of host cells for a time sufficient for at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral vector or phage life cycles. In some embodiments, the time sufficient for one phage life cycle is about 10 minutes.

Therefore, in some embodiments, the time of the entire evolution procedure is about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 50 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about two weeks, about 3 weeks, about 4 weeks, or about 5 weeks.

For example, in some embodiments, a PACE apparatus is provided, comprising a lagoon of about 100 ml, or about 1 l volume, wherein the lagoon is connected to a turbidostat of about 0.5 l, 1 l, or 3 l volume, and to a vessel comprising an inducer for a mutagenesis plasmid, for example, arabinose, wherein the lagoon and the turbidostat comprise a suspension culture of E. coli cells at a concentration of about 5×10⁸cells/ml. In some embodiments, the flow of cells through the lagoon is regulated to about 3 lagoon volumes per hour. In some embodiments, cells are removed from the lagoon by continuous pumping, for example, by using a waste needle set at a height of the lagoon vessel that corresponds to a desired volume of fluid (e.g., about 100 ml, in the lagoon. In some embodiments, the host cells are E. coli cells comprising the F′ plasmid, for example, cells of the genotype F′ proA⁺B⁺ Δ(lacIZY) zzfi:Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻. In some embodiments, the selection phage comprises an M13 genome, in which the pIII-encoding region, or a part thereof, has been replaced with a gene of interest, for example, a coding region that is driven by a wild-type phage promoter. In some embodiments, the host cells comprise an accessory plasmid in which a gene encoding a protein required for the generation of infectious phage particles, for example, M13 pIII, is expressed from a conditional promoter as described in more detail elsewhere herein. In some embodiments, the host cells further comprise a mutagenesis plasmid, for example, a mutagenesis plasmid expressing a mutagenesis-promoting protein from an inducible promoter, such as an arabinose-inducible promoter. In some embodiments the apparatus is set up to provide fresh media to the turbidostat for the generation of a flow of cells of about 2-4 lagoon volumes per hour for about 3-7 days.

Vectors and Reagents

The invention provides viral vectors for the inventive continuous evolution processes. In some embodiments, phage vectors for phage-assisted continuous evolution are provided. In some embodiments, a selection phage is provided that comprises a phage genome deficient in at least one gene required for the generation of infectious phage particles and a gene of interest to be evolved.

For example, in some embodiments, the selection phage comprises an M13 phage genome deficient in a gene required for the generation of infectious M13 phage particles, for example, a full-length gIII. In some embodiments, the selection phage comprises a phage genome providing all other phage functions required for the phage life cycle except the gene required for generation of infectious phage particles. In some such embodiments, an M13 selection phage is provided that comprises a gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and a gX gene, but not a full-length gIII. In some embodiments, the selection phage comprises a 3′-fragment of gIII, but no full-length gIII. The 3′-end of gIII comprises a promoter (see FIG. 16) and retaining this promoter activity is beneficial, in some embodiments, for an increased expression of gVI, which is immediately downstream of the gIII 3′-promoter, or a more balanced (wild-type phage-like) ratio of expression levels of the phage genes in the host cell, which, in turn, can lead to more efficient phage production. In some embodiments, the 3′-fragment of gIII gene comprises the 3′-gIII promoter sequence. In some embodiments, the 3′-fragment of gIII comprises the last 180 bp, the last 150 bp, the last 125 bp, the last 100 bp, the last 50 bp, or the last 25 bp of gIII. In some embodiments, the 3′-fragment of gIII comprises the last 180 bp of gIII.

M13 selection phage is provided that comprises a gene of interest in the phage genome, for example, inserted downstream of the gVIII 3′-terminator and upstream of the gIII-3′-promoter. In some embodiments, an M13 selection phage is provided that comprises a multiple cloning site for cloning a gene of interest into the phage genome, for example, a multiple cloning site (MCS) inserted downstream of the gVIII 3′-terminator and upstream of the gIII-3′-promoter.

Some aspects of this invention provide a vector system for continuous evolution procedures, comprising of a viral vector, for example, a selection phage, and a matching accessory plasmid. In some embodiments, a vector system for phage-based continuous directed evolution is provided that comprises (a) a selection phage comprising a gene of interest to be evolved, wherein the phage genome is deficient in a gene required to generate infectious phage; and (b) an accessory plasmid comprising the gene required to generate infectious phage particle under the control of a conditional promoter, wherein the conditional promoter is activated by a function of a gene product encoded by the gene of interest.

In some embodiments, the selection phage is an M13 phage as described herein. For example, in some embodiments, the selection phage comprises an M13 genome including all genes required for the generation of phage particles, for example, gI, gII, gIV, gV, gVI, gVII, gVIII, gIX, and gX gene, but not a full-length gIII gene. In some embodiments, the selection phage genome comprises an F1 or an M13 origin of replication. In some embodiments, the selection phage genome comprises a 3′-fragment of gIII gene. In some embodiments, the selection phage comprises a multiple cloning site upstream of the gIII 3′-promoter and downstream of the gVIII 3′-terminator.

In some embodiments, the selection phage does not comprise a full length gVI. GVI is similarly required for infection as gIII and, thus, can be used in a similar fashion for selection as described for gIII herein. However, it was found that continuous expression of pIII renders some host cells resistant to infection by M13. Accordingly, it is desirable that pIII is produced only after infection. This can be achieved by providing a gene encoding pIII under the control of an inducible promoter, for example, an arabinose-inducible promoter as described herein, and providing the inducer in the lagoon, where infection takes place, but not in the turbidostat, or otherwise before infection takes place. In some embodiments, multiple genes required for the generation of infectious phage are removed from the selection phage genome, for example, gIII and gVI, and provided by the host cell, for example, in an accessory plasmid as described herein.

The vector system may further comprise a helper phage, wherein the selection phage does not comprise all genes required for the generation of phage particles, and wherein the helper phage complements the genome of the selection phage, so that the helper phage genome and the selection phage genome together comprise at least one functional copy of all genes required for the generation of phage particles, but are deficient in at least one gene required for the generation of infectious phage particles.

In some embodiments, the accessory plasmid of the vector system comprises an expression cassette comprising the gene required for the generation of infectious phage under the control of a conditional promoter. In some embodiments, the accessory plasmid of the vector system comprises a gene encoding pIII under the control of a conditional promoter the activity of which is dependent on a function of a product of the gene of interest.

In some embodiments, the vector system further comprises a mutagenesis plasmid, for example, an arabinose-inducible mutagenesis plasmid as described herein.

In some embodiments, the vector system further comprises a helper plasmid providing expression constructs of any phage gene not comprised in the phage genome of the selection phage or in the accessory plasmid.

Evolved Products

Some aspects of this invention provide evolved products of continuous evolution processes described herein. For example, some embodiments provide a modified T7 RNA polymerase (T7RNAP) having an altered substrate specificity and/or an increased transcriptional activity as compared to wild type T7RNAP. In some embodiments, a modified T7RNAP is provided that initiates transcription from a T3 promoter. In some embodiments, the modified T7RNAP exhibits an at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold greater transcription rate from its native T7 promoter than the wild-type enzyme. In some embodiments, the modified T7RNAP exhibits an at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, or at least 50-fold greater transcription rate from a non-native T7RNAP promoter than the wild-type enzyme. In some embodiments, the non-native T7RNAP promoter is a T3 promoter.

Host Cells

Some aspects of this invention relate to host cells for continuous evolution processes as described herein. In some embodiments, a host cell is provided that comprises at least one viral gene encoding a protein required for the generation of infectious viral particles under the control of a conditional promoter. For example, some embodiments provide host cells for phage-assisted continuous evolution processes, wherein the host cell comprises an accessory plasmid comprising a gene required for the generation of infectious phage particles, for example, M13 gIII, under the control of a conditional promoter, as described herein. In some embodiments, the host cells comprise the gene required for the generation of infectious viral particles under the control of a conditional promoter inserted into their genome, or as a cosmid, phagemid, or an artificial chromosome. In some embodiments, the host cell is a bacterial cell, for example, a bacterial cell amenable to M13 infection, such as certain E. coli cells. For M13 PACE, the host E. coli cells need to express the F-factor, for example, from an F′ plasmid. Suitable F′ E. coli cell lines and strains are known to those of skill in the art, and include, for example, the F′ proA⁺B⁺ Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻ cells described herein.

In some embodiments, the host cell provided further comprises an expression construct comprising a gene encoding a mutagenesis-inducing protein, for example, a mutagenesis plasmid comprising a pBAD promoter, as described elsewhere herein.

Kits

Some aspects of this invention provide kits for continuous evolution as described herein. In some embodiments, a kit provided herein comprises a selection phage and a suitable accessory plasmid, as described in more detail elsewhere herein. In some embodiments, the kit may also comprise a helper phage or phagemid, a mutagenesis plasmid, a mutagen, an inducer, and/or a suitable host cell.

The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention.

General Methods.

All equipment, reagents, suppliers, and relevant catalog numbers are detailed in Table 2. All PCR reactions were performed with HotStart Phusion II polymerase. Water was purified using a MilliQ water purification system (Millipore, Billerica Mass.).

DNA Cloning.

All vectors were constructed by isothermal assembly cloning. [44] 5× isothermal assembly buffer contained 3 mL 1 M Tris-HCl pH 7.5, 300 μL 1 M MgCl₂, 600 μL 10 mM dNTPs, 300 μL 1 M dithiothreitol, 1.5 g PEG-8000, 20 mg NAD, and H₂O to 6 mL. Individual 320 μL aliquots were frozen at −20° C. Isothermal assembly master mix was prepared by mixing 320 μL 5× buffer with 1 μL T5 Exonuclease, 20 μL Phusion polymerase, 160 μL Taq DNA ligase, and H₂O to 700 μL. Individual 15 μL aliquots in PCR tubes were frozen at −20° C. DNA fragments to be assembled were PCR-amplified using oligonucleotide primers designed to ensure between 30 and 40 base pairs of overlap homology with each adjacent fragment. DpnI was added directly to the PCR reactions to remove template DNA, followed by PCR cleanup with MinElute columns according to the manufacturer's protocol. Fragments were assembled by mixing equimolar amounts in a total 5 μL with 15 μL isothermal assembly master mix and incubating at 50° C. for 1 hr. Assembly mixtures were directly transformed into NEBTurbo competent cells by heat shock or purified by MinElute columns as described prior to electroporation.

Plasmids.

T7 RNAP-dependent accessory plasmids (APs) contained, in order, a strong rrnB terminator, the promoter of interest, a desired ribosome binding site, gene III, the bla gene conferring carbenicillin resistance, and either the pUC or SC101 origin of replication. For selection stringency assays, RBS A=5′-AAGGAGGTAACTCATAGTG-3′ (SEQ ID NO: 12), RBS B=5′-AAGGAAATAACTCATAGTG3′ (SEQ ID NO: 13), and RBS C=5′-AAGAAAATAACTCATAGTG-3′ (SEQ ID NO: 14), where underlined bases represent the start codon of gene III. Reporter plasmids were identical to SC101 accessory plasmids except for the replacement of gene III by full-length lacZ. T7 RNAP selection phage (SP) was constructed by replacing all but the last 180 bp of gene III with the gene encoding T7 RNAP in VCSM13 helper phage. The mutagenesis plasmid (MP) consisted of dnaQ926, umuD′, umuC, and recA730 under control of the araC operon. Expression plasmids used for quantification assays consisted of the cloD13 origin of replication, aadA, and the wild-type gene III promoter and RBS driving expression of the evolved T7 RNAP variant. All plasmids used in this work are described in Table 1. Vector maps of representative plasmids are shown in FIG. 9.

Bacterial Strains.

All DNA cloning was performed with Mach1 cells or NEB Turbo cells. Early discrete infection assays and PACE experiments were performed with PirPlus DH10βFDOT cells.

Plaque assays and PACE experiments with T7 RNAP were performed using E. coli S109 cells derived from DH10B by replacement of the proBA locus with the pir116 allele, as previously described. [45] To our knowledge, this modification was not required for PACE experiments with T7 RNAP. Similarly, the lacI cassette was deleted from the F plasmid and from the chromosome to enable mutagenesis assays. S109 cells were rendered F⁺ by conjugation with ER2738. The complete genotype of the resulting strain is F′ proA⁺B⁺ Δ(lacIZY) zzE:Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻.

Discrete Infection Assays.

Discrete (non-continuous) infectivity assays were performed by cotransforming phage bearing antibiotic resistance genes with the appropriate accessory plasmid into competent cells to generate phage donors. For two-hybrid and recombinase experiments, phage producing cells contained gIII-deleted helper phage, accessory plasmid, and selection phagemid.

Colonies were picked and grown overnight in 2×YT media with both antibiotics. 2 μL donor cells were mixed with 198 μL F⁺ recipient cells in mid-exponential phase containing an antibiotic resistance gene not found in the donor. Mixtures were incubated at 37° C. for 1.5 hours and 20 μL was spread on plates containing the donor and the recipient antibiotics. Infection was quantified by the number of resulting colonies after incubation at 37° C. overnight. For plaque assays, phage DNA was transformed into electrocompetent cells containing the appropriate accessory plasmid and recovered for 1 hr at 37° C. Serial dilutions were mixed with 300 μL F+ recipient cells grown to exponential phase in 14 mL Falcon culture tubes and incubated at 37° C. for 15 minutes. 3 mL top agar (7 g/L from LB broth base) at 50° C. was added to each tube, briefly vortexed, and poured onto minimal agar plates incubated at 37° C. for 8 hours or overnight to generate plaques.

Turbidostat Assembly.

Assembly followed the schematic in FIG. 2b. Turbidostats were constructed from BioProbe flasks on magnetic stir plates. Each flask was equipped with a TruCell2 cell density meter held in a GL32 probe holder with compression fitting. GL45 and GL32 septa pierced with needles, transferred media to and from the turbidostat via an 8-channel peristaltic pump with Tygon tubing. A needle set at the desired turbidostat volume level pumped excess cells to the waste container.

A 0.2 μm filter attached to a 14-gauge needle piercing the septum vented the turbidostat vessel. A two-way valve controlled media flow to the turbidostat, connected such that a closed valve state returned the media to source. The valve opened and closed in response to TruCell2 4-20 mA output processed by a digital panel meter programmed with the desired set point. Panel meters were unlocked according to the instruction manual and programmed to the following settings: Input dc_A, Setup 30_10, Config 00000, Filtr 11009, dec.pt ddd.dd, lo in 00.400, lo rd 004.00, hi in 02.000, hi rd 020.00, Alset 00036, deu1h 000.01, deu2h 000.01. The digital panel meter, adapter, and valve were connected with a solderless breadboard according to the diagram shown in FIG. 15. Lagoons consisted of 100 mL Pyrex bottles with GL45 septa pierced with needles for fluid delivery, a 0.2 μm filterterminated vent line, and a magnetic stir bar. Excess lagoon volume was continually pumped to waste via a waste needle set at the desired lagoon volume.

Media Preparation.

Each 20 L media carboy received 140 g anhydrous potassium phosphate dibasic, 40 g potassium phosphate monobasic, 20 g ammonium sulfate, and 20 mL Tween 80 in 20 L H₂O.

Carboys were loosely capped with Polyvent filling/venting closures with an autoclavable 0.2 μm filter fastened to the venting port. Media was autoclaved until visibly boiling (typically 120 min at 30 psi, 121° C.) and allowed to cool overnight. Media supplement was prepared from 90 g glucose, 10 g sodium citrate, 0.25 g anhydrous magnesium sulfate, 10 g casamino acids, 0.15 g tetracycline-HCl, 0.6 g carbenicillin, 0.6 g spectinomycin, and 0.5 g (L)-leucine, dissolved in 500 mL H₂O, and filtered with a Nalgene 500 mL filtration unit. 500 mL media supplement was added to each carboy under conditions that minimize the risk of media contamination (in the case of the reported experiments, immediately following 1 hour of germicidal UV irradiation).

Sterilization and Cell Culture.

The autoclavable components of a turbidostat apparatus include the BioProbe flask, TruCell2 probe, needles, vent filter, and tubing. All such components were autoclaved fully assembled except for tubing, which was connected while hot. Upon equilibrating to ambient temperature, the peristaltic pump responsible for media addition and waste removal was started with the valve opened until the desired volume was reached. The TruCell2 probe was connected to its transmitter and zeroed. Turbidostats were seeded with 100 μL of an overnight culture of host cells. Turbidostats and lagoon cultures were grown at 37° C.

Cell Density Calibration.

Serial dilution plating was used to generate a calibration curve to determine TruCell2 output and panel meter setting corresponding to the desired cell density. For these experiments, both panel meter alarms were programmed to open the valve at 6.80 mA. Cells were pumped from the turbidostat to the lagoons via peristaltic pumps with silicone (platinum) two-stop tubing. Calibration curves relating pump speed in rpm to volumetric flow rate were determined experimentally with a timer and graduated cylinder for each tubing size.

PACE Experiments.

Turbidostats and lagoons were assembled as described above. Upon the turbidostat reaching the desired set point of 6.80 mA corresponding to 5×10⁸cells/mL, lagoons were connected to turbidostats, waste needles were set at the desired volume, and lagoon pumps were set to a flow rate corresponding to the desired dilution rate. Each lagoon was seeded with 100 μL of an overnight culture producing selection phage. To induced elevated mutagenesis, 10% filter-sterilized (L)-arabinose was delivered by a separate peristaltic pump to each lagoon requiring enhanced mutagenesis to a final concentration of 1%. Lagoon aliquots were taken by sampling lagoon waste lines at the luer lock just after the peristaltic pump. Individual clones were isolated by plaque assay or amplified by PCR, assembled into a T7 RNAP activity assay plasmid, and transformed into cells containing a lacZ reporter plasmid. Active clones were picked by blue/white screening.

Selection Phage Optimization.

T7 RNAP was subcloned into VCSM13 helper phage encoding kanamycin resistance, generating HP-T7RNAP A, used in the discrete infection assay shown in FIG. 3a.

To ensure that improvements to the phage genome did not interfere with the evolution of T7 RNAP, HP-T7RNAP A was propagated in a lagoon fed by S109 host cells containing AP-T7 A and DP-QUR and supplemented with arabinose for 48 hours at 2.0 volumes/hour. Individual plaques were isolated and their T7 RNAP genes sequenced. One plaque contained only a single point mutation in T7 RNAP, P314T, and was chosen as the preoptimized selection phage for T7 RNAP evolution. Sequencing of the rest of the selection phage revealed numerous changes relative to the parental VCSM13. Notably, the entire p15a-Kan^Rcassette inserted into the intergenic (IG) region to create VCSM13 was perfectly deleted to reconstitute the wild-type M13 IG region. Other changes included N79S, F286S, and 1360T mutations in gIV, a K249R mutation in gII, three silent mutations back to the corresponding M13 base, two other silent mutations, and the deletion of one thymine residue in the terminator before gIII, possibly increasing the expression of T7 RNAP. These regional patterns of variation parallel those observed by Husimi in more extensive filamentous phage evolution experiments. [46] This evolved phage, designated SP-T7RNAP P314T, was used as the starting selection phage for all subsequent PACE experiments.

Mutagenesis Assays.

The lacI gene was cloned into VCSM13 between the p15a origin and kan^rto generate VCSM13-lacI. A turbidostat was grown to a set point equivalent to 5×10⁸cells/mL with S109 cells containing the mutagenesis plasmid (MP). Lagoons were seeded with 10 μL VCSM13-lacI and run at 2.5 volumes/hour for 3 hours. One lagoon was supplemented with 10% arabinose to a final concentration of 1%, while the other was not. Aliquots were removed after 3 hours and each was used to infect a 100-fold greater volume of recipient cell culture of S109 cells containing a reporter plasmid conferring carbenicillin and spectinomycin resistance with a lacI binding site (lacO) capable of repressing spectinomycin resistance. Mixtures were incubated for 1.5 hr at 37° C., spread on 2×YT plates containing spectinomycin, kanamycin, and carbenicillin, and incubated at 37° C. overnight.

Colonies were counted for induced and uninduced lagoons to estimate the fold increase in mutagenesis.

Seventy-two (72) colonies were sequenced to determine the frequencies of all transitions and transversions within mutated lacI genes. The results are shown in FIG. 11. All sequenced colonies contained at least one mutation capable of inactivating repressor function.

In Vivo T7 RNAP Activity Assays.

Overnight cultures of S109 cells grown in 2×YT containing reporter plasmid and expression vector were diluted 4-fold in fresh 2×YT media. 20 μL of the diluted culture was mixed with 80 μL Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 M BME, pH 7.0) in Falcon Microtest 96-well OptiLux assay plates and the absorbance at 595 nm was measured using a Spectra M5 plate reader. 25 μL of 1 mg/mL methylumbelliferyl-beta-(D)-galactopyranoside was added to each well and the time recorded. Plates were incubated at 30° C. and fluorescence was measured at 360/460 nm on a Spectra M5 plate reader. Plates were measured at multiple time points to avoid saturation of the spectrophotometer or consumption of the substrate, depending on the activity level of the T7 RNAP enzyme being assayed. MUG fluorescence units were calculated as previously described. [47]

T7 RNAP Protein Purification.

T7 RNAP variants were cloned into pT7-911Q, a His-tagged T7 RNAP expression vector. [48] Overnight cultures grown at 30° C. were diluted 1:500 in LB broth containing 50 μg/mL carbenicillin and 2% glucose. Upon reaching OD₆₀₀=˜0.5, cultures were centrifuged at 4000 g for 5 minutes and resuspended in LB broth with 0.4 mM IPTG and 50 μg/mL carbenicillin. Cultures were grown for 4 hours at 30° C., spun at 8000 g for 6 minutes, and the pellet was frozen overnight. Binding buffer consisted of 50 mM Tris, 300 mM NaCl, 5% glycerol, 5 mM beta-mercaptoethanol, and 10 mM imidazole at pH 8.0. Wash buffer was composed of 50 mM Tris, 800 mM NaCl, 5% glycerol, 5 mM beta-mercaptoethanol, and 20 mM imidazole at pH 8.0. Elution buffer was equivalent to wash buffer with 500 mM imidazole. Pellets from 25 mL culture were resuspended in 1 mL wash buffer and cells were lysed by sonication while kept on ice using a Misonix CL4 sonicator at maximal microtip power for 45 seconds in 1-second bursts. Cell debris was spun down at 20,000 g for 15 minutes at 4° C. Ni-NTA spin columns were equilibrated with 500 μL binding buffer and spun at 800 g for 2 minutes. Lysate supernatant was loaded onto each column and spun at 300 g for 5 minutes. Columns were washed twice with 500 μL wash buffer, spinning at 800 g for each, then eluted twice with 250 μL elution buffer. Proteins were dialyzed into 20 mM Tris, 100 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, pH 8.0, and concentrated using Amicon Ultra-0.5 30K concentration columns.

In Vitro T7 RNAP Activity Assays.

Purified T7 RNAP variant concentrations were determined by Bradford assay and then by Coomassie stain on a 4-12% NuPage gel. Templates were prepared by PCR amplification of 150 bp fragments of the reporter plasmids used for in vivo assays including the promoter and the start of the lacZ gene. Templates were purified by MinElute spin column.

Transcription reactions were performed in 1×RNA polymerase buffer consisting of 40 mM Tris-HCl, 6 mM MgCl₂, 10 mM dithiothreitol, 2 mM spermidine pH 7.9 with 1 mM rNTPs, 1 ng template DNA, purified polymerase variant, and 2 mCi [α-³²P]ATP. Reactions were incubated at 37° C. for 20 minutes, mixed with an equivalent volume of loading dye consisting of 7 M urea, 178 mM Tris-Cl, 178 mM H₃BO₃, 4 mM EDTA and 0.002% bromophenol blue, then electrophoresed on Criterion 5% or 10% TBE-urea denaturing gels. To control for increased signal from [α-³²P]ATP in the iA₆promoter and ensure transcripts fully entered the gel, iA₆and wt T7 RNAs were transcribed from double-stranded templates of sequence 5′-TAATACGACTCACTATAGGGAGAGCCACCACCACCACCACCACCA-3′ (SEQ ID NO: 15) and 5′-TAATACGACTCACTATAAAAAAAGCCACCACCACCACCACCACCA-3′ (SEQ ID NO: 16), digested with T1 ribonuclease to remove the first seven bases, and electrophoresed on Criterion 15% TBE-urea gels. Phosphor screens were exposed and imaged on a Typhoon Trio phosphorimager. Bands corresponding to transcription products were quantified with ImageJ software.

RACE Analysis.

In vitro transcription reactions with purified T7 RNAP variants were performed as described above but without the addition of any radioactive nucleotide using polymerase variant C6-80.9. DNA oligonucleotides of sequence 5′-TAATACGACTCACTATACCC-3 (SEQ ID NO: 17)′ and 5′-CCCACCCAAAAAAAAAAAAAAAGGGGGGTATAGTGAGTCGTATTA-3′ (SEQ ID NO: 18) formed the iC₆template, and 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO: 19) and 5′-CCCACCCAAAAAAAAAAAAAAATCTCCCTATAGTGAGTCGTATTA-3′ (SEQ ID NO: 20) formed the wild-type template. Each 200 μL transcription reaction was treated with 2 μL calf intestinal phosphatase (CIP) for 1 hour at 37° C., extracted with phenol-chloroform twice to remove enzymes, and precipitated with ethanol. Pellets were resuspended in 1×DNase Turbo buffer with 5 μL DNase in a total volume of 100 μL, incubated for 2 hours at 37° C., extracted with phenol-chloroform twice, and precipitated with ethanol. 5 μL of purified transcript was mixed with 1 μL T4 polynucleotide kinase (PNK) in 1×PNK buffer and incubated at 37° C. for 1 hour. 1 μL of treated RNA was ligated to 30 ng RNA adapter of sequence 5′-GCUGAUGGCGAUGAAUGAACACUGCGUUUGCUGGCUUUGAUGAAA-3′ (SEQ ID NO: 21) with T4 RNA Ligase in 1×RNA Ligase Buffer at 37° C. for 1 hour. 1 μL of ligated RNA was reverse transcribed by mixing with 1 mM dNTPs and a complementary DNA primer of sequence 5′-CCCCCAAACCCCCAAAAAAAAAACCCACCCAAAAAAAAAAAA-3′ (SEQ ID NO: 22) at a final concentration of 5 μM at 65° C. for 15 minutes followed by 10 U/μL SuperScript III reverse transcriptase in 1×RT buffer, 5 mM MgCl₂, and 10 mM dithiothreitol at 50° C. for 1 hour. Enzymes were denatured at 85° C. for 5 minutes followed by amplification with nested PCR reactions using primers 5′-CGATCCGAACGCAGCATTTACGCTGATGGCGATGAATGAACACTG-3′ (SEQ ID NO: 23) and 5′-CCCCCAAACCCCCAAAAAAAAAACCCACCCAAAAAAAAAAAA-3′ (SEQ ID NO: 24) followed by 5′-AACGCCAGCAACGCGAATAAGAGAATACATCGATCCGAACGCAGC-3′ (SEQ ID NO: 25) and 5′-GCTAGTTATTGCTCAGCGGAAAAAAAAAAAAACCCCCAAACCCCC-3′ (SEQ ID NO: 26). PCR products were subcloned with TOPO TA kits. Individual colonies were picked and sequenced using the M13 reverse primer.

Calculation of Phage Generation Time During PACE.

The phage generation time was estimated using a steady-state replication model in which phage fitness is constant. One phage generation encompasses an infected cell exiting the lagoon, one of its progeny phage infecting an incoming cell, and that infected cell exiting the lagoon. To determine the average time required for one of the progeny phage to infect an incoming cell, it was assumed that no progeny phage have yet infected an incoming cell and that cells entering the lagoon are instantaneously infected due to the high phage (5×10¹⁰mL⁻¹) and cell (5×10⁸mL⁻¹) concentrations relative to the adsorption coefficient of filamentous phage (3×10⁻¹¹mL⁻¹min⁻¹phage⁻¹cell⁻¹). [49] Because lagoons operate in a steady state during most of a PACE experiment, the total phage and cell concentration does not change over time. Therefore, the progeny of a single cell exiting the lagoon infects exactly one cell entering the lagoon, on average, to achieve population replacement. A number of incoming cells equal to the total cell population will enter before one of the progeny phage achieves infection. Consequently, the average time from instantaneous cell exit to infection of a new cell by a progeny phage is 1/(dilution rate).

According to the dilution equation, solutes wash out at a rate of dC/dt=−(dilution rate)*(concentration). This gives a washout equation of C(t)=c1*exp[−(dilution rate)*time]. This washout equation may be used as the probability density function prob [time] for the probability of a given cell washing out at a specific time after it enters the lagoon. To ensure that the probability of all currently resident cells washing out given infinite time is 1, the dilution rate was set to c1=1/(dilution rate). To calculate the probability <t> for a single cell, the integral [t*prob[t], {t, 0, infinity}] was calculated. The integral of [time*(dilution rate)*exp[−(dilution rate)*time], {t, 0, infinity}]=1/(dilution rate). Therefore, the average time from infection to washout is also equal to 1/(dilution rate).

Summing the two halves of the phage replicative cycle, the average phage generation time in our model is equal to 2/(dilution rate). At a dilution rate of 2.0 volumes/hour, as in the evolution towards T3 promoter recognition, the average phage undergoes 24 generations per 24 hours. At 2.5 volumes/hour, as in the initiation site evolution experiments, the average phage undergoes 30 generations per 24 hours. For the maximum observed dilution rate of 3.2 volumes/hour, the average phage undergoes just over 38 generations per 24 hours. In all cases the fastest replicating 1% of phage have undergone a significantly greater number of generations per 24 hours.

Phage-Assisted Continuous Evolution (PACE)

A scheme was devised that exploits the continuous culture and selection of the M13 filamentous bacteriophage [15,16] (commonly used in phage display [17]) to enable the continuous directed evolution of proteins or nucleic acids. In phage-assisted continuous evolution (PACE), E. coli host cells continuously flow through a fixed-volume “lagoon” containing an actively replicating population of phage DNA vectors encoding the gene(s) of interest (FIG. 1).

The average residence time of host cells in the lagoon is less than the time required for E. coli replication. As a result, mutations accumulate only in the evolving population of phage vectors since they are the only replicating DNA in the lagoon. The mutation of host cells in the lagoon should therefore have minimal impact on the outcome of the selection over many rounds of phage replication, and mutagenesis conditions are not limited to those that preserve E. coli viability.

PACE achieves continuous selection by linking the desired activity to the production of infectious progeny phage containing the evolving gene(s). Phage infection requires protein III (pIII; encoded by gene III), which mediates F pilus binding and TolA-dependent host cell entry. [18,19] Phage lacking pIII are ˜10⁸-fold less infectious than wild-type phage. [20] Crucially, the production of infectious phage scales with increasing levels of pIII over concentrations spanning two orders of magnitude. [21] To couple pIII production and thus phage infectivity to the activity of interest, gene III was deleted from the phage vector and inserted it into an “accessory plasmid” (AP) present in the E. coli host cells (see FIG. 9 for plasmid maps). The production of pIII from the AP is dependent upon the activity of the evolving gene(s) on the phage vector and in the absence of this activity is insufficient to support the production of infectious phage. Phage vectors able to induce sufficient production of pIII from the AP will therefore propagate and persist in the continuously diluted lagoon, while phage vectors unable to induce pIII will be washed out over time (FIG. 2). Because pIII expression level determines the rate of infectious phage production, [21] the progeny of a phage encoding a mutant gene that results in a higher level of pIII production will infect a larger share of host cells relative to the progeny of a phage encoding a less active gene. Mutants with higher activity will therefore experience a selective advantage until pIII levels are sufficient for maximal infectious phage release.

Due to the brevity of the phage life cycle (phage production begins ˜10 minutes postinfection), [22] PACE can mediate many generations of phage replication under selective conditions in a single day. Activity-dependent phage vectors were observed that tolerate lagoon flow rates up to 3.2 volumes per hour (FIG. 10), corresponding to ˜115 population doublings and an average of ˜38 phage generations per 24 hours. Because each lagoon is simply a vessel maintained at constant volume, multiple lagoons can evolve genes in parallel, with each 100 mL lagoon containing ˜5×10¹⁰host cells selectively replicating active phage variants.

Importantly, PACE requires little or no intervention during evolution and obviates the need to create DNA libraries, transform cells, extract genes, or perform DNA cloning steps during each round.

Linkage of Functions to pIII

Production

In principle, PACE is capable of evolving any gene that can be linked to pIII production in E. coli, including activities that influence pIII function at the transcriptional, translational, or post-translational levels. Because a wide variety of functions including DNA binding, RNA binding, protein binding, bond-forming catalysis, and a variety of enzyme activities have been linked to the expression of a reporter protein through n-hybrid [23-25] and other conditional transcription strategies, PACE can be applied to the evolution of many different activities of interest. As examples, protein-protein binding, recombinase activity, and RNA polymerase activity were successfully linked to phage infectivity in discrete infection assays by creating variants of the AP that associate each of these activities with pIII production (FIG. 3). PACE applies optimal evolutionary pressure when pIII levels are above the minimal threshold required to prevent total phage loss, but below the amount needed to produce maximally infectious phage. This window can be shifted in at least two ways to accommodate the current activity level of the encoded genes. It was observed that selection stringency can be tuned by varying the copy number of the AP or by altering the ribosome binding site (RBS) [26] of gene III to modulate the efficiency with which gene III is transcribed or translated (FIG. 4).

Inducible Enhanced Mutagenesis

Although the basal mutation rate of replicating filamentous phage is only 5.3×10⁻⁷substitutions/bp, [27] the large number of phage-vectors infecting cells in a 100 mL lagoon (˜5×10¹⁰) ensures the presence of all possible single point mutants in any phage vector of viable size at any instance. To enhance the rate of gene diversification in PACE in a controllable manner, an inducible mutagenesis plasmid (MP) was constructed that elevates the error rate during DNA replication and facilitates the use of chemical mutagens. The addition of arabinose to the lagoon induces expression of the DNA polymerase dominant negative proofreading subunit dnaQ926 [28] and the production of the specialized repair DNA polymerase pol V. [29] The increase in mutagenesis was measured by continuously propagating phage encoding the lac repressor (lacI) in lagoons with or without arabinose and selecting for loss-of-function mutants after three hours of continuous propagation.

Full induction with 0.8% arabinose increased the apparent mutagenesis rate of the lad gene by ˜100-fold, inducing all possible transitions and transversions (FIG. 11).

This increase in mutagenesis rate has a significant impact on the nature of PACE gene libraries. For a 1 kb target gene, an enhanced mutation rate of ˜5×10⁻⁵substitutions/bp yields 2.5×10⁹mutations in 5×10¹⁰phage after one generation, sufficient to generate all possible single and double mutants and a significant number of triple mutants. Because the arabinose-induced mutagenesis rate enables a 100 mL lagoon to exhaustively sample all possible double mutants of any starting phage vector, single-mutation fitness valleys can be traversed during PACE.

Continuous Evolution of T7 RNA Polymerase: Promoter Recognition

The DNA-dependent RNA polymerase of bacteriophage T7 (T7 RNAP) is widely used to transcribe RNA in vitro and to drive protein expression in cells. T7 RNAP is highly specific for its promoter sequence (TAATACGACTCACTATA, SEQ ID NO: 27), and exhibits virtually no detectable activity on the consensus promoter of the related bacteriophage T3 (AATTAACCCTCACTAAA, SEQ ID NO: 28). The nucleotide at position −11 (G in the T7 promoter; C in the T3 promoter) is known to play an especially important role in T7 RNAP promoter recognition, but the known N748D mutation in T7 RNAP that confers recognition of a C base at position −11 still exhibits <5% activity on the T3 promoter. [30] Despite decades of study and several attempts to engineer the specificity of T7 RNAP towards other promoters including that of T3, a mutant T7 RNAP capable of recognizing the T3 promoter has not been previously reported. [31-34] PACE was challenged to evolve T7 RNAP recognition of the complete T3 promoter.

First PACE was used to optimize a phage vector containing wild-type T7 RNAP in place of gIII to remove potential interference from evolutionary improvements to the phage vector rather than to T7 RNAP. The vector was continuously propagated for three days at a lagoon flow rate of 2.0 volumes per hour on host cells containing an AP with the wild-type T7 promoter driving gill expression. A single plaque presumed to represent vector-optimized T7 RNAP-expressing phage contained a single mutation (P314T) and was used as the starting point for T7 RNAP evolution. It was confirmed that the activity of the P314T mutant does not measurably differ from that of wild-type T7 RNAP (FIG. 12). It was examined whether this optimized T7 polymerase-expressing phage was able to propagate with an AP containing the T3 promoter under the weakest possible selection stringency. Phage vectors containing T7 RNAP survived continuous dilution with host cells containing the T7 promoter AP but failed to propagate and were washed out of the lagoon when diluted instead with host cells containing the T3 promoter AP. Consistent with the lack of reported T7 RNAP variants capable of recognizing the T3 promoter, these results suggest that no immediately accessible mutants possess sufficient T3 promoter activity to propagate continuously on the complete T3 promoter.

It was then tested whether T7 RNAP mutants with T3 promoter recognition activity could be evolved by using PACE to first evolve T7 RNAP mutants, to recognize a hybrid promoter with the T7 promoter base at the important −11 position but all other positions changed to their T3 promoter counterparts, then simply switching to host cells harboring the complete T3 promoter AP. T7 RNAP phage vectors were propagated on the hybrid T7/T3 promoter AP in two initially identical lagoons in parallel at a constant flow rate of 2.0 volumes per hour for 60 hours, then on the T3 promoter AP for 48 hours, and finally on a high-stringency, single copy T3 promoter AP for 84 hours (FIG. 5a).

In both lagoons phage persisted after 7.5 days total of PACE, surviving a net dilution of 10¹⁷⁰fold, the equivalent of ˜550 phage population doublings and ˜200 rounds of evolution by the average phage. Phage vectors were isolated, sequenced, and characterized from each lagoon after 48, 108, and 192 hours. Up to eight non-silent mutations in a single T7 RNAP gene were observed at 48 hours, up to ten non-silent mutations per clone were observed at 108 hours, and up to 11 non-silent mutations per clone were observed at 192 hours. In addition, several mutations in promoters driving T7 RNAP expression were observed in multiple clones although none of these became predominant during the course of the evolution.

Protein-encoding regions (without upstream promoter sequences) of evolved mutant T7 RNAP genes were cloned into assay plasmids that quantitatively link transcriptional activity to beta-galactosidase expression in cells. [35] It was defined that 100% activity is that of the starting T7 RNAP on the T7 promoter. The starting T7 RNAP exhibited undetectable (≦3%) levels of activity on the T3 promoter in these cell-based assays. At 48 hours, before the switch to the full T3 promoter, PACE-evolved variants of T7 RNAP exhibited up to 20% activity on the T3 promoter (≧6-fold improvement; FIGS. 5b and 5c), with a similar increase of at least 6-fold over the starting enzyme on the T7 promoter. All clones isolated from plaques after 108 hours of PACE exhibited at least 200% activity on the T3 promoter, corresponding to a ≧65-fold increase over the starting activity (FIGS. 5b and 5c). Clones isolated after 192 hours of PACE exhibited at least 300% activity on the T3 promoter, ≧100-fold higher than the starting activity (FIGS. 5b and 5c). The T3 promoter recognition activity of the most active clone improved by ≧300-fold over the 7.5-day course of the experiment, representing a shift from undetectable activity in cells to nearly ten times the activity of wild type T7 RNAP on the wild type T7 promoter.

Despite the lack of an explicit negative selection against wild-type T7 promoter recognition, some clones isolated after 108 and 192 hours of PACE exhibited lower T7 promoter recognition activity than their ancestors, presumably through genetic drift in the absence of selection pressure to recognize the T7 promoter (FIGS. 5b and 5c). One clone, L2-108.2, exhibited 270% of the target activity on the T3 promoter and only 100% activity on the T7 promoter, representing a net ≧100-fold change in specificity compared with wild-type T7 RNAP. These results collectively establish the ability of PACE to very rapidly evolve large changes in enzyme activity and specificity with minimal intervention by the researcher.

Several evolved T7 RNAP mutants were also purified and assayed in vitro using standard radioactive nucleotide incorporation assays. [36] Purified T7 RNAP mutants exhibited activity levels consistent with the cell-based assays of up to 90-fold over the starting T3 promoter recognition activity (FIG. 5d). In vitro activities did not as dramatically exceed the activity of the wild-type T7 RNAP on the T7 promoter, suggesting that evolved improvements in T7 RNAP activity in cells include improvements in features such as expression level, polymerase folding, or stability in addition to improvements in substrate binding or catalytic rate. Mutational analysis of the PACE-evolved T7 RNAP genes revealed basic structure activity relationships among mutant clones (FIG. 6). The N748D mutation known to enable recognition of the T3 base at the −11 position appeared in isolates of both lagoons within 48 hours after shifting to the full T3 promoter (FIG. 6). Two other known mutations observed during PACE are E222K, a known specificity broadener, [33] and K98R, a change to the corresponding amino acid in T3 RNAP that directly contacts the −15 and −16 bases altered in the T3 promoter. These two mutations were never observed in the same clone, suggesting mutual exclusivity or similar effects on RNAP specificity.

Interestingly, the evolutionary dynamics of the two initially identical lagoons differed significantly (FIG. 6). Within 24 hours, lagoon 1 acquired a predominant suite of mutations consisting of 14M, G175R, E222K, and G542V and changed little thereafter until acquiring N748D following exposure to the full T3 promoter. In contrast, lagoon 2 accessed these mutations more slowly before an entirely different set of mutations, including K98R, became predominant at 96 hours, only to be displaced in turn by the same five mutations observed in lagoon 1. The presence of several mutations unique to lagoon 2 throughout the experiment suggests that lagoon cross-contamination did not occur. The distinct evolutionary trajectories of the two lagoons prior to their ultimate convergence upon a set of common mutations highlight the ability to PACE to rapidly discover multiple viable pathways to a target activity in parallel experiments.

Continuous Evolution of T7 RNA Polymerase: Transcript Initiation

T7 RNAP is highly specific for initiation with GTP, [37-39] significantly limiting its usefulness for the in vitro transcription of RNAs that begin with other nucleotides. As initiation has been described as a mechanistically challenging step in transcription, [40] it was attempted to use PACE to evolve T7 RNAP variants capable of initiating transcription with other nucleotides. T7 RNAP is known to initiate with GTP up to several bases downstream of the +1 position if the template is devoid of early guanines in the coding strand. [41] Therefore, accessory plasmids were constructed in which positions +1 through +6 of the gene III transcript were CCCCCC (iC₆) or AAAAAA (iA₆), as well as a library consisting of randomized bases at those positions (iN₆).

To apply selection pressure towards a pool of variants capable of more promiscuous initiation with non-G bases, vector-optimized T7 RNAP phage was propagated on host cells carrying the iN₆AP library. After 48 hours of selecting for initiation at random bases, it was switched to host cells containing a high-copy iC₆accessory for 16 hours, and finally to cells carrying a low-copy iC₆accessory for 16 hours (FIG. 7a). At a dilution rate of 2.5 volumes/hour, phage survived a total dilution of 10⁸⁹, or ˜100 phage generations. Ten sequenced clones from the end of the experiment contained up to 16 total mutations (up to 7 non-silent). It was observed that wild-type T7 RNAP retains a surprising amount of activity [41] on the iC₆promoter (50% of that on the wild-type promoter). All PACE-evolved clones exhibited at least 80% activity on an iC₆promoter in cell-based assays, with the most active clone exhibiting 280% activity, an improvement of 5.3-fold (FIG. 7b). In vitro transcription assays of purified mutant T7 RNAP proteins confirmed the 50% activity of wild-type T7 RNAP on the iC₆promoter, with evolved variants exhibiting improvements of up to 2.3-fold (FIG. 7c). As in the case of the proteins evolved to recognize the T3 promoter, it can be inferred that the somewhat higher cellular activities of iC₆-evolved variants are due to improvements in protein folding and stability.

Mutational analysis revealed that the H524N and A827V mutations were conserved among all 12 sequenced clones, presumably arising very early during PACE. In addition, all clones contained either K577M or both C125R and S128R. Several clones contained all five mutations, including the most active clone assayed, suggesting that both observed sets of mutations increase activity and are mutually compatible. Of the five mutations, only C125R and S128R are predicted to be capable of contacting and stabilizing the newly initiated RNA, although A827V is in close proximity to the active site.

Although the requirements of functional pIII or LacZ protein in the above experiments ensure that evolved RNA polymerases retain overall sequence fidelity, it is in principle possible that transcription is initiated with non-templated nucleotides rather than the base programmed by the template. To test this possibility, RNA transcripts generated by evolved polymerase C₆-80.9 on both the iC₆template and the wild-type template (which initiates transcription with GGGAGA) were sequenced by the RACE method. For both wild-type and iC₆templates, transcripts were initiated with the template-directed bases rather than nontemplated nucleotides. (FIG. 13) As a final evolutionary challenge, PACE was used to rapidly evolve polymerases that can initiate transcription using an iA₆promoter. Starting phage was propagated in host cells with a high-copy iA₆AP for 24 hours, followed by a 30:70 high-copy:single-copy mixture of host cells for 12 hours (FIG. 8a). At a dilution rate of 2.5 volumes per hour, phage survived a total dilution of 10⁴⁰and experienced an average of ˜45 rounds of replication under selective conditions. Of six sequenced clones isolated after only 36 hours of evolution, all exhibited at least four non-silent mutations, with one clone possessing seven non-silent mutations.

Starting from undetectable initial activity (<3%), all six evolved clones exhibited at least 270% activity on the iA₆promoter in cell-based assays, representing an improvement of at least 33-fold over the starting activity, while retaining 120% activity on the wild-type promoter (FIG. 8b). The most active evolved clone exhibited 430% activity on the iA₆promoter, corresponding to ≧51-fold improvement in activity. In vitro transcription with purified variants yielded activities on the iA₆promoter that are comparable to that of wild type T7 RNAP on the wild-type promoter, an improvement of at least 28-fold in activity (FIG. 8c).

All six characterized iA₆-derived clones emerging from PACE contained K93T, S397R, and S684Y mutations, while three of the six also contained S228A. Three of the four mutated residues are predicted to be distant from the nascent RNA and the active site, suggesting that distal changes to the promoter-binding regions of T7 RNAP may result in conformational changes in the active site that better accommodate initiation with adenosine.

In contrast, residue 397 is thought to directly contact the nascent RNA strand, [42] suggesting a direct role for S397R in allowing efficient initiation of iA₆transcripts.

Continuous Negative Selection

Selecting solely for activity on a new target substrate without counter-selection against activity on non-target substrates is likely to result in enzymes with broadened, rather than altered, specificity. Negative selection strategies that exert evolutionary pressure against undesired activities, including poor specificity, are useful to avoid or minimize undesired off-target activity, and allow the evolution of enzymes with high specificity, by linking undesired activities to the inhibition of phage propagation (FIG. 17).

A negative selection strategy was developed in which the selection stringency can be fine-tuned, and in which selection is dependent on the ratio of desired and undesired activities, rather than the absolute level of the undesired activity. A dominant negative variant of pIII was identified that satisfies both of these criteria. This variant (“pIII-neg”) comprises the two N-terminal domains of pIII fused to a truncated, termination-incompetent C-terminal domain as described in Bennett, N.J.; Rakonjac, J., Unlocking of the filamentous bacteriophage virion during infection is mediated by the C domain of pIII. Journal of Molecular Biology 2006, 356 (2), 266-73; the entire contents of which are incorporated herein by reference. This C-terminal domain allows incorporation into a progeny phage but is unable to catalyze the unlocking of the particle for entry during infection, rendering the progeny phage noninfectious. Furthermore, when this variant is present in a phage containing wild-type pIII (˜five total pIII molecules are incorporated into each phage), pIII-neg exhibits a dominant negative effect by blocking the unlocking cascade normally catalyzed by wild-type pIII (FIG. 17).

Because this negative selection agent is a protein, pIII-neg, the stringency of counterselection can be tuned by varying the strength of the RBS upstream of the pIII-neg coding region, thereby allowing for a defined modulation of the level of penalty incurred for a given degree of undesired activity. Furthermore, wild-type pIII and pIII-neg compete for incorporation into a progeny phage particle, which makes the potency of inhibition sensitive to the ratio of the desired and undesired activities, rather than just the absolute level of undesired activity.

A dual selection accessory plasmid was constructed (FIG. 17). The dual selection plasmid comprises a wild-type pIII coding sequence placed under the control of an inducible promoter (P_Tet), and a pIII-neg coding sequence placed under the control of a different inducible promoter (P_Lac). The two pIII transgenes were expressed at different levels in cells producing phage particles (low wt pIII/low pIII-neg; high wt pIII/low pIII-neg; high wt pIII/high pIII-neg; and low wt pIII/high pIII-neg, respectively), and infectivity of the resulting phage was evaluated (FIG. 17). The infectivity of the resulting progeny phage reflects the ratio of wildtype pIII expression to pIII-neg expression, rather than merely reflecting expression levels of either pIII variant alone.

In order to obtain a T7 RNA polymerase that specifically recognizes a T3 promoter but has minimal or no activity on a T7 promoter, an accessory plasmid is constructed in which a T3 promoter sequence drives expression of a wt pIII coding sequence (positive selection), and a T7 promoter drives expression of pIII-neg. A PACE procedure is then performed as described herein, starting with wild type T7 RNA polymerase, or an evolved T7 RNA polymerase exhibiting transcriptional activity on a T3 promoter. It is expected that the evolution products obtained from this dual selection PACE procedure exhibit strong transcriptional activity on a T3 promoter, and little or no activity on a T7 promoter.

Continuous Selection for Protease Activity

For continuous evolution of proteases with tailor-made protein cleavage specificities, a selection strategy is devised that links target proteolysis to phage propagation. This linkage is achieved, in some embodiments, by using a previously reported lambda repressor-based strategy that links protease activity with gene expression by rendering the lambda repressor protein sensitive to site-specific proteolysis (see, e.g., Sices, H. J.; Kristie, T. M., A genetic screen for the isolation and characterization of site-specific proteases. Proc Natl Acad Sci USA 1998, 95 (6), 2828-33; and Sices, H. J.; Leusink, M. D.; Pacheco, A.; Kristie, T. M., Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease. AIDS Res Hum Retroviruses 2001, 17 (13), 1249-55, the entire contents of each of which are incorporated herein by reference). Lambda repressor (cI) contains an N-terminal DNA binding domain and a C-terminal dimerization domain connected by a long flexible linker. Each cI dimer binds cooperatively to operator sites within the lambda pR promoter and represses transcription upon binding. This high-affinity DNA binding requires cI dimerization since free monomeric N-terminal domains bind only weakly to their operator half-sites. Therefore, the separation of the N- and C-terminal domains of cI by proteolysis results in decreased binding to the operator sites and derepresssion of the pR promoter, which in turn can drive pIII production (FIG. 18). The highly cooperative nature of cI binding ensures that promoter activation is very responsive to relatively modest decreases in the concentration of intact cI.

A pIII expression construct was generated in which a pR promoter (containing cI binding sites) drives expression of pIII and luciferase (FIG. 18). The expression construct was expressed in cells expressing cI comprising a TEV protease cleavage site (ENLYFQ(G/S), SEQ ID NO: 29) within the interdomain linker. The cI was expressed from an inducible IPTG promoter. The expression of cI was modulated by the addition of various concentrations of IPTG. Increased levels of IPTG resulted in increasing repression of the pR promoter, while simultaneous expression of TEV protease from an arabinose-inducible promoter resulted in a dramatic (>100-fold) stimulation of gene expression (FIG. 18). This transcriptional activation depended on both the catalytic activity of TEV (the active site mutant TEV C151A does not stimulate expression) and the proper recognition site within the cI linker (mutation of the P1 position glutamine to alanine abolishes activation by WT TEV protease).

These results establish a strong linkage between protease cleavage activity and an increase in pIII expression that is useful for PACE protease selection. The finding that high concentrations of cI keep pR substantially repressed even in the presence of TEV protease activity (FIG. 18), allows for tunable protease selection stringency.

A negative selection for protease activity targeting similar, but off-target recognition sites, is desirable in some embodiments. Further, a negative selection can be used to ensure that the positive selection applies pressure for cleavage of the intended recognition site within the cI linker, and not elsewhere within cI. A negative selection is established by installing into the AP an analogous cI-repressed expression cassette that results in pIII-neg expression (FIG. 19). In this cassette, undesired protease recognition site(s) are incorporated into the linker of a cI variant (“cI-neg”) that is orthogonal to wild-type cI in both dimerization specificity and DNA-binding specificity. Such orthogonal cI variants are known from related lambda-family phages (see, e.g., Wharton, R. P.; Ptashne, M., Changing the binding specificity of a repressor by redesigning an alphahelix. Nature 1985, 316 (6029), 601-5; and Wharton, R. P.; Ptashne, M., A new-specificity mutant of 434 repressor that defines an amino acid-base pair contact. Nature 1987, 326 (6116), 888-91, the entire contents of each of which are incorporated herein by reference). These cI-neg variants are highly similar to cI, apart from DNA sequence specificity differences required to impart orthogonality. When localized upstream of pIII-neg (through use of a pR promoter variant with cognate binding sites for cI-neg), the cleavage of cI-neg will result in expression of pIII-neg and inhibition of phage production (FIG. 19). Thus, a protease variant that cleaves at the undesired recognition site or within an unintended region of the repressor that is common to both cI and cI-neg (most of the repressor molecule) will produce fewer infectious progeny phage than variants with more specific activity against the desired recognition site.

All four of the evolved T7 RNAP variants assayed in vitro exhibit the ability to initiate transcription using iC₆, iA₆, and wild-type templates with efficiencies comparable to that of wild-type T7 RNAP initiating with the wild-type template (FIG. 14), and therefore may represent improved, more general T7 RNAP variants for routine in vitro transcription.

Each of the three PACE experiments yielded T7 RNAP variants with significantly higher in vivo activities than that of wild-type T7 RNA polymerase transcribing the wild-type T7 promoter. PACE also revealed different combinations of mutations that achieve this effect on the T7, T3, iC₆, and iA₆promoters, indicating the existence of multiple accessible evolutionary paths conferring increased activity in cells. That none of these paths were taken by wild-type T7 phage suggests that there was no selection pressure for more efficient polymerization during the evolution of T7 bacteriophage, that retaining specificity requires lower overall activity, or a combination of both possibilities.

By rendering bacteriophage infection dependent upon a molecular activity of interest, the efficiency of the phage life cycle was harnessed to enable the continuous directed evolution of proteins or, in principle, other gene-encoded molecules without the need for in vitro library creation, gene harvesting, or gene manipulation steps. The PACE system can be assembled entirely from a modest collection of commercially available equipment (listed in Table 2) and does not require the manufacture of any specialized components.

PACE proceeds at a rate of several dozen rounds of mutation, selection, and gene replication per day, representing roughly a 100-fold increase over most current protein evolution methods. These capabilities may be particularly well-suited to address problems or questions in molecular evolution that require hundreds to thousands of generations, or the execution of multiple evolution experiments in parallel. More generally, PACE represents the integration and manipulation of many protein and nucleic acid components in a living system to enable the rapid generation of biomolecules with new activities, a significant goal of synthetic biology.

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All publications, patents and sequence database entries mentioned herein, including those items listed above, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.