A method for visual continuous spatially directed evolution

Abstract

To provide a method of directed evolution in visible continuous space.SOLUTION: Directed evolution (also called laboratory evolution) is an effective technological means that can generate biomolecules with specific functions by controlling biological evolutionary processes. A host grows and moves in a solid culture space, the host has an evolving foreign target gene, the host itself contains genetic elements that support the evolution of the target gene, and the target gene is related to the growth and movement of the host. In the process of the growth and movement of the host, evolutionary products are obtained by forming and screening different spatial distribution patterns in the solid culture space.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to a Chinese patent application filed with the China Patent Office on December 27, 2017, entitled "Method for visual continuous spatially directed evolution" and bearing patent application number 201711446362.3, the entire contents of which are incorporated herein by reference.

[0002] The present invention is in the field of directed evolution screening, and specifically relates to a method for visual continuous spatial directed evolution. [Background technology]

[0003] Directed evolution (also known as laboratory evolution) is an effective technological tool that can generate biomolecules with specific functions by controlling the biological evolution process. The generated biomolecules are widely used in many fields, including industrial production, biotechnology, and drug development. David R. Liu's laboratory at Harvard University developed a phage-assisted continuous evolution system (PACE, Phage Assisted Continuous Evolution). The system mainly consists of three modules: LWS, CCP, and IMP. In the phage module (LWS), the gIII gene in the phage M13 gene required for packaging and infection of host bacteria is excised and the gene for the biomolecule to be evolved is inserted. Phages with excised gIII are unable to generate progeny phages by infecting hosts. In the mutagenesis module (IMP), arabinose is used as an inducer to induce the expression of DNAQ926, dam, and seqA, preventing the excision of inappropriate bases introduced by the polymerase during DNA replication and increasing the mutation rate. The arabinose-induced IMP can increase the phage mutation rate several hundred-fold. The auxiliary module (CCP) contains the gIII gene used by the phage to infect and propagate in host cells. The expression of gIII on the CCP is correlated with the biological activity of the target gene being evolved on the LWS (e.g., if an RNA polymerase needs to evolve in a specific direction, the expression of gIII can be controlled by a promoter that matches the evolution direction). This determines whether each mutant LWS can generate progeny phage LWS with infectious activity.

[0004] When a phage carrying the wild-type target gene to be evolved in the evolution pool infects a host cell, it injects its genetic material, wild-type LWS, into the host bacterium and replicates it via the host bacterium's replication system. Furthermore, in the presence of arabinose induction, the expression of the IMP DNAQ926, dam, and seqA genes in the host cell induces mutations in the LWS. If the mutations acquired by the LWS (i.e., mutations in the target gene on the LWS) can promote the expression of the gIII protein, infectious progeny phage can be generated. The resulting progeny mutant LWSe is secreted outside the host bacterium and infects new host bacteria for further replication and proliferation. As a result, the LWSe mutant that can promote gIII expression continues to proliferate and increase in number, while the wild-type LWS and mutant LWS that cannot promote gIII expression are unable to secrete progeny phage, propagate, or increase in number. At this point, if new host bacterial cultures are added to the evolutionary pool at a constant rate while the original culture is discharged, wild-type LWS and its mutants that are unable to promote gill expression or have low expression ability will be quickly eluted, while LWSe mutants that can efficiently promote gill expression will be retained until the end.

[0005] The system is relatively large and requires continuous fed-batch cultivation using a series of automated fed-batch fermenters equipped with a highly sensitive real-time monitoring system. Because various evolution products are mixed and diluted in the fermenter, direct detection and separation are difficult. The system has drawbacks, such as the consumption of large amounts of reagents, the high cost of the fermentation equipment, and the complicated evolution process. Furthermore, the system has the drawback of low throughput because it can only evolve one target gene at a time.

[0006] In view of this, the following invention is proposed. Summary of the Invention

[0007] To solve the problems of the prior art, such as the difficulty of sample detection and separation, low throughput, high cost, and complicated evolution procedures, the present invention provides a visual continuous spatially directed evolution method. This method allows direct evolution in a solid culture space, such as the surface of a solid culture plate, without the need for a liquid fed-batch culture device. Furthermore, the spatial morphology and distribution of infection spots formed during the evolution process can be observed with the naked eye, allowing the evolution effect to be understood. This method does not require a real-time monitoring device, is simple to operate, has high throughput, and allows multiple groups of evolution experiments to be conducted simultaneously.

[0008] To achieve the above object, the present invention employs the following technical means.

[0009] A method for visual continuous spatially directed evolution, wherein a host is grown and moved in a solid culture space, said host carrying an exogenous target gene to be evolved, said host itself containing genetic elements that assist in the evolution of said target gene, said target gene being linked to the growth and movement of said host; The growth and migration of the host creates different spatial distribution patterns in the solid culture space, and the evolution products are obtained by screening against these patterns.

[0010] In the visual continuous spatially directed evolution method (SPACE) of the present invention, the system includes a target gene to be evolved and a host, where the host contains genetic elements that assist the evolution of the target gene, and the target gene is associated with the host. The target gene evolves as the host grows and moves in a solid culture space, and after evolution, various spatial distribution patterns that can be recognized by the naked eye are formed, and the evolution products are selected in this state.

[0011] The entire evolution and screening process spans a two-dimensional plane or three-dimensional space, with different evolutionary products distributed in different regions of the plane and unable to mix with each other. The different activities of each product create distinct spatial distribution pattern images in localized areas that can be recognized by the naked eye. As evolution continues, these images expand further. As evolutionary products emerge, images can be generated at the corresponding locations. Even if the amount of product is extremely small, the image does not become faint or unclear. Based on the state of the image, evolutionary products that meet the needs can be directly selected and isolated. The system is simple to operate, inexpensive, and requires no special equipment. It allows one person to conduct evolution experiments on multiple groups at once, and can achieve high-throughput directed evolution of target genes.

[0012] In some embodiments of the present invention, the target gene is present in the host genome, a plasmid, or a parasite corresponding to the host, and the target gene is inserted into the host genome by genetic recombination, and a host containing the target gene to be evolved can be obtained by introducing a plasmid into the host or by invading the host with a parasite.

[0013] In an embodiment of the present invention, the parasite comprises any one of a phage (bacteriophage), a cyanophage, an animal or plant virus, a fungal virus, a mycoplasma, a chlamydia, and a bacterium.

[0014] In an embodiment of the invention, the parasite is a phage; The host is the natural host bacterium of a non-defective strain of said phage, a strain obtained by genetic recombination of a non-defective strain of said phage with the natural host bacterium, a non-native host bacterium obtained by genetic modification and with reduced susceptibility (a non-native host bacterium that acquires susceptibility only after genetic modification), The host includes Escherichia coli, Pasteurella, Shigella, Pseudomonas, Xanthomonas, Salmonella, Staphylococcus aureus, and modified strains whose susceptibility is changed by genetic recombination; Preferably, the host is Escherichia coli carrying a fertility factor F, the phage is a temperate phage, a virulent phage, or a chronically infecting phage; The phages include filamentous phages, T4 phages, T7 phages, λ phages, P1 phages, P2 phages, P22 phages, φX174 phages, and SP6 phages; Preferably, the filamentous phage includes M13 filamentous phage and f1 filamentous phage.

[0015] In a preferred embodiment of the present invention, the phage is an M13 phage, the gill gene required for packaging and infection of a host bacterium is excised, and a genetic element that assists the evolution of a target gene, such as a helper plasmid, contains the gill gene. Phages are generally capable of invading host bacteria and replicating DNA. However, in the absence of a specific helper plasmid, they are unable to package infectious progeny.

[0016] In an embodiment of the present invention, the target genes are a combination of one or more protein coding and non-coding genes.

[0017] In an embodiment of the present invention, the target gene is selected from one or more of a T7 RNA polymerase gene, a protease gene, a cellulase gene, a fluorescent protein gene, and a density-sensing gene.

[0018] In an embodiment of the present invention, the genetic element that assists the evolution of the target gene is a mutagenesis plasmid, which enhances or induces the expression of the target gene before and after evolution, respectively.

[0019] Preferably, the mutagenesis plasmid contains a mutagenesis gene, which is at least one of the DNAQ gene mutant DNAQ926 gene in which the amino acids at positions 12 and 14 are mutated to Ala, the deoxyadenosine methylase dam gene, the hemimethylated GATC-binding protein seqA gene, the activation-induced cytosine deaminase gene AID, the uracil-DNA glycosylase inhibitor gene Ugi in phage PBS2, and the transcriptional repressor emrR.

[0020] Mutagenizing genes can increase the mutation rate of genetic information during transfer processes such as replication and transcription. Mutagenizing plasmids (IMPs) can be induced and expressed in the same way (e.g., induced and controlled by the psp promoter). In different embodiments, when multiple mutagenizing plasmids (IMPs), such as IMP1, IMP2, and IMP3, exist, these mutagenizing plasmids can be the same, and the target genes before and after evolution can be expressed differently. In this case, IMP and IMP2 represent different IMPs.

[0021] In an embodiment of the present invention, the solid culture space includes a two-dimensional planar culture structure and a three-dimensional spatial culture structure.

[0022] In an embodiment of the present invention, the vertical movement and evolution of the solid culture space is maintained continuity by a periodically formed casting solid culture system.

[0023] In an embodiment of the present invention, the directed evolution is high-throughput evolution.

[0024] In embodiments of the present invention, the high-throughput evolution can be achieved by multiple solid culture spaces or various locations of the solid culture space.

[0025] In an embodiment of the present invention, the target gene is linked to the growth and movement of the host via a helper plasmid, which comprises at least a first helper plasmid, the first helper plasmid being helper plasmid CCP1 or helper plasmid CCP2, the nucleic acid sequence of helper plasmid CCP1 being as set forth in SEQ ID NO:3 (SEQ ID NO:3), and the nucleic acid sequence of helper plasmid CCP2 being as set forth in SEQ ID NO:4 (SEQ ID NO:4).

[0026] The helper plasmid CCP1 or helper plasmid CCP2 supports low-level replication and proliferation before phage evolution, and after evolution, when the activity of the target gene increases, the helper plasmid CCP1 or helper plasmid CCP2 supports phage evolution to a higher level of replication and proliferation.

[0027] In an embodiment of the present invention, the helper plasmid further comprises a second helper plasmid, wherein the second helper plasmid is helper plasmid CCP3 or helper plasmid CCP4, and the nucleic acid sequence of helper plasmid CCP3 is as shown in SEQ ID NO:5 (SEQ ID NO:5).

[0028] The helper plasmids CCP3 and CCP4 are functionally defective and therefore unable to support the propagation of pre-evolved phages.

[0029] Specifically, the host bacterium S1 possesses IMP1 and CCP1 and is capable of growing and moving on a culture plate. During migration, the host bacterium S1 comes into contact with the phage LWS and evolves with LWS until the phage LWSe, carrying the evolved target gene, is generated. Because the phage LWS has a growth defect, the pre-evolved LWS is able to infect and replicate at low levels due to background expression of CCP1. The function of at least a portion of the evolved target gene is linked to the function of the genetic element CCP2, which supports the propagation of the evolved phage LWSe. Therefore, CCP1 allows LWSe to efficiently carry out infectious replication in S1. When phage replication is efficient, host bacterial growth is suppressed, resulting in the formation of a clear infection spot with few bacteria in the LWSe-infected area, which is visible to the naked eye. The infection spot allows direct analysis of the effects of evolution.

[0030] In an embodiment of the present invention, the host bacterium further includes host bacterium S2 or host bacterium S3, which grows and migrates on a culture plate. Host bacterium S2 first comes into contact with evolved phage LWSe during migration and evolves with LWSe until phage LWSeN, which carries the evolved target gene, is produced. Host bacterium S2 includes helper plasmid CCP2, which supports the propagation of evolved phage LWSe or LWSeN, helper plasmid CCP3, which suppresses the propagation of naive phage LWS, and mutagenesis plasmid IMP2. The function of at least a portion of the evolved target gene is related to the function of the genetic elements on CCP2, which support the propagation of evolved phage LWSe or LWSeN. The function of the genetic elements on CCP3 and CCP4 is related to the function of the naive target gene, and the genetic elements on CCP3 and CCP4 are functionally defective and cannot support the propagation of naive phage LWS. This further reduces the wild-type activity of the target gene on phage LWSeN. If the wild-type activity of the target gene in phage LWSeN is sufficiently low, the use of CCP2 in S2 allows efficient infectious replication. Efficient phage infectious replication inhibits the growth of the host bacterium, resulting in the formation of a clear infection spot with few bacteria visible to the naked eye in the phage LWSeN infection area. This infection spot allows direct analysis of the effects of evolution. The host bacterium S3 contains the helper plasmid CCP2, which supports the growth of the evolved phage LWSe or LWSeN, the helper plasmid CCP4, which inhibits the growth of the original phage LWS, and the mutagenesis plasmid IMP2. CCP4 is a high-copy plasmid and exhibits a high level of defective genetic elements. In other words, CCP4 is an enhanced form of CCP3. Substituting S3 bacteria for S2 bacteria allows further evolution of phage LWSe. Similarly, the CCP2 plasmid has a lower copy number than CCP1, and the background expression of the low-copy CCP2 is low, so it cannot support the replication of the original phage LWS, providing stronger selective pressure for evolution.

[0031] In an embodiment of the invention, the method further comprises, when the host bacterium has migrated to the edge of the plate, moving the host bacterium and evolved phage to a next plate to continue evolution.

[0032] In an embodiment of the present invention, the directed evolution is carried out by alternating between different hosts, and the genetic elements contained in the latter hosts that support phage propagation include a helper plasmid that supports the propagation of the evolved phage and a helper plasmid that suppresses the propagation of the naive phage.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) The method for visual continuous spatial directed evolution provided by the present invention has spatial attributes. The entire evolution process is carried out in two-dimensional or three-dimensional space, with space serving not only as a support platform but also as a selective pressure for evolution. Even small amounts of evolutionary products directly form spatial distribution patterns such as plaques at predetermined locations. Therefore, by monitoring the size of infection spots, the number of plaques, or reporter genes formed during evolution in real time, the activity of the target gene and the proliferation activity of the phage can be understood, and the evolutionary products can be directly recognized and isolated. Therefore, no liquid fed-batch culture device or real-time monitoring device is required.

[0035] (2) When the method for visual continuous spatially directed evolution according to the present invention is adopted, evolutionary results appearing at different spatial locations are fixed at those locations and can be directly separated without being affected by dilution and mixing with other components. Furthermore, the signals of evolutionary results appearing at different spatial locations are amplified along the evolutionary direction, thereby improving the evolutionary effect and achieving higher detection sensitivity.

[0036] (3) The method of visual continuous spatial directed evolution according to the present invention allows for simple evolutionary manipulation, high throughput, and allows for evolutionary experiments of multiple groups at once. [Brief explanation of the drawings]

[0037] In order to more clearly describe the technical matters of the embodiments of the present invention or the prior art, the drawings relating to the embodiments or the prior art will be briefly described below.

[0038] [Figure 1] FIG. 1 shows the effect of host bacteria migration and infection spotting in an example of the present invention. [Figure 2] FIG. 1 illustrates the principle of continuous spatial directed evolution according to an embodiment of the present invention. [Figure 3] FIG. 1 illustrates a model of continuous spatial directed evolution according to an embodiment of the present invention. [Figure 4] FIG. 1 shows the results of the first SPACE positive screening evolution in Example 4 of the present invention. [Figure 5] FIG. 1 shows the results of multiple rounds of SPACE evolution in Examples 5 and 6 of the present invention. [Figure 6] 1 is a bar graph showing the effect of SPACE evolution induced by IMP3 and the number of different phages in Experimental Example 1 of the present invention. [Figure 7] 1 is a bar graph showing the infection transfer effect of T7 phage and the number of phages in Experimental Example 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0039] The technical aspects of the present invention will be described in detail below with reference to specific examples. However, as is well known, the following examples are merely illustrative and do not limit the technical scope of the present invention. Unless specific conditions are specified in the following examples, they may be carried out under conventional conditions or conditions recommended by the manufacturer. Unless the manufacturer of the reagents or equipment used in the present invention is specified, they are conventional products available on the market.

[0040] In the method of visual continuous spatially directed evolution provided by the present invention, when phages and host bacteria are mixed and coated on a plate, there is a difference in growth between host bacteria infected with M13 phage and those not infected, resulting in the formation of plaques that can be recognized by the naked eye. Reporter genes have problems related to specificity, sensitivity, and operability. The present invention can visualize SPACE without using a reporter gene by directly using the spots formed during the infection process as an indicator signal for SPACE.

[0041] Similar to the plaque formation experiment, host bacteria are inoculated in the center of a culture plate, and wild phage are inoculated at the three corners around the host bacteria (see Figure 1a). During this process, the host bacteria grow on the plate and migrate toward the edge of the plate. As they move, they come into contact with phages, become infected, produce progeny phages, and continue to migrate toward the outer edge. Because the growth of infected host bacteria is slow, while the host bacteria in uninfected areas remain intact, a V-shaped infection spot with few bacteria is formed in the infected area, which is visible to the naked eye (see Figure 1b). The clear infection spot indicates phage infection, and the transparency and size of the clear spot can be used to indicate the phage's infection activity.

[0042] The core design principle of SPACE, invented by the inventors of the present invention based on their experience studying spatial evolution systems, is shown in Figures 2 and 3. When a host bacterium carrying the mutagenesis plasmid IMP and the helper plasmid CCP is infected with the phage LWS carrying a target gene, the phage genome undergoes replication and mutation. If the mutant progeny LWS can activate the expression of the gIII gene on the CCP, a progeny phage LWSe with infectious activity is generated, and the next round of infectious evolution begins. If activation is not possible, a defective progeny phage without infectious activity is generated. As evolution progresses, the number of LWSe continues to increase, while the number of wild-type LWS phage and progeny phage with infectious activity is not increased or increases slightly. The gene of interest (GOI) on the LWS in the figures represents the target gene to be evolved.

[0043] Host bacteria containing the CCP and IMP are inoculated in the center of a culture plate, and phage LWS is inoculated at the three corners around the host bacteria. As the host bacteria grow and divide on the plate, they move toward the edge of the plate using their flagella, and at specific locations come into contact with phage LWS that were inoculated in advance at that location. The phage LWS invades the host bacteria and moves forward while carried by the host bacteria. Initially, the phage LWS cannot promote the expression of gIII on the CCP, and can only grow at a low level due to background expression of the CCP. Since the phage growth level is very low at this time, it has little effect on the host bacteria. Infected and uninfected host bacteria grow and divide at roughly the same rate, with no visible morphological differences. At the same time, the low-level growth rate of the phage LWS is slower than the growth and movement rate of the host bacteria, so inefficient phage LWS does not move. They are quickly overtaken by the host bacteria.

[0044] As phage LWSe propagates, it undergoes mutational evolution via IMP until it evolves into phage LWSe, which can efficiently promote CCP expression. At this time, phage LWSe directly promotes CCP, and highly efficient proliferation results in the continuous generation of progeny phage, which continuously infect the moving bacteria. The efficient proliferation of phage LWSe disrupts the growth of the host bacteria, slowing bacterial growth. Therefore, a clear zone is formed at the designated location of phage LWSe formed by evolution, where host bacteria grow slowly and there are fewer bacteria. As evolution progresses and the bacteria infected by phage LWSe continue to move, this clear zone continues to expand in the direction of movement, eventually forming a V-shaped infection spot on the plate that is visible to the naked eye. The closer to the outside of the infection spot, the longer the evolution time and the more obvious the evolution effect. The desired evolved phage LWSe can be obtained from the infected spot.

[0045] As shown in Figures 2 and 3, the phage-assisted continuous spatial directed evolution system according to one embodiment of the present invention includes two parts: positive screening (a) and negative screening (b). In a possible embodiment of the present invention, the positive screening (a) alone can achieve the goal of evolving the target gene to be evolved into the evolved target gene. Therefore, the negative screening (b) can be regarded as a technical solution obtained by further improving the directed evolution system of the present invention. In practical application, the positive screening (a) part realizes a qualitative change from the "target gene to be evolved" to the "evolved target gene," and the negative screening (b) part realizes a quantitative change that further enhances the function of the "evolved target gene."

[0046] In the present invention, the so-called "target gene to be evolved" (GOI) can be the same as the "target gene before evolution." That is, a target gene that has not undergone evolutionary mutations through the systems and methods of the present invention can also be called a "wild-type gene." The so-called "target gene after evolution" is the opposite of the "target gene to be evolved." The "target gene after evolution" has a new evolved function, but may also retain its original function. The original function may even be completely lost. In the present invention, if a target gene has a new function through evolution, it can be called an "evolved target gene," regardless of whether the original function is present or not.

[0047] It should be noted that the symbols used herein, such as LWS, CCP, IMP, and S, are merely exemplary symbolic representations. Other names may be used for the objects to which these symbols are attached. For example, phage LWS may be referred to as an unevolved phage, and phage LWSe may be referred to as an evolved phage, or may be collectively referred to as evolved phages. Phage LWSeN may also be referred to as a further evolved phage. The number N (e.g., 1, 2, 3, or other combinations of letters and numbers) may refer to the number of passages, the number of evolutions, or evolution under different conditions. Host bacterium S1 is referred to as the first host bacterium, host bacterium S2 as the second host bacterium, and host bacterium S3 as the third host bacterium. Helper plasmid CCP1 is referred to as the first helper plasmid, helper plasmid CCP2 as the second helper plasmid, helper plasmid CCP3 as the third helper plasmid, and helper plasmid CCP4 as the fourth helper plasmid. Mutagenesis plasmid IMP1 can also be referred to as the first mutagenesis plasmid, and mutagenesis plasmid IMP2 can also be referred to as the second mutagenesis plasmid. Mutagenesis plasmid IMP3 can also be referred to as the third mutagenesis plasmid. Plasmids CCP1 and CCP2, which support the growth of evolved phage LWSe, can also be referred to collectively as positive screen CCPs. Similarly, plasmids CCP3 and CCP4, which suppress the growth of unevolved phage LWS, can also be referred to collectively as negative screen CCPs. Plasmids IMP1, IMP2, and IMP3 used to generate mutations are collectively referred to as IMPs.

[0048] The host bacteria of the present invention may include not only the host bacteria S1, S2, and S3 but also other host bacteria. The host bacteria S1, S2, and S3 of the present invention do not mean that they are of different "bacterial species," but that the helper plasmids or mutagenesis plasmids contained in the strains are different. In the present invention, the host bacteria S1, S2, and S3 may be bacteria obtained by introducing different plasmids into the same bacterial species, such as E. coli carrying the F factor.

[0049] As shown in Figures 2 and 3, a phage-assisted continuous spatially directed evolution system according to one embodiment of the present invention includes a phage LWS carrying a target gene to be evolved. The phage LWS has a growth defect. A "growth defect" refers to a defect in a specific function of the phage life cycle, such as packaging and / or infection of a host bacterium. This function can be caused by a mutation in a related gene. Different phages have different genes responsible for functions such as packaging and / or infection of a host bacterium. For example, in one embodiment of the present invention, the phage LWS is an M13 phage, and the gIII gene used for packaging and infection of a host bacterium is deleted, preventing normal packaging and infection of the host bacterium. As is well known, any phage similar to the above phage can be used as the phage LWS of the present invention, and the present invention is not limited to the M13 phage.

[0050] In the directed evolution system shown in Figures 2 and 3, the host bacterium S1 contains the helper plasmid CCP1 and the mutagenesis plasmid IMP1, which support the propagation of evolved LWSe. The background expression of CCP1 allows low-level infection and proliferation of the pre-evolved LWSe. The helper plasmids CCP1 and CCP2 support the propagation of evolved LWSe by linking the function of at least a portion of the evolved target gene with the function of the genetic elements on CCP1 and CCP2 that support the propagation of evolved LWSe. The term "at least a portion" refers not only to linking the function of a portion of the evolved target gene with the function of the genetic elements that support the propagation of evolved LWSe, but also to linking the function of that portion with the function of the genetic elements that support the propagation of pre-evolved LWSe. For example, in one embodiment of the present invention, the target gene before evolution refers to the T7 RNA polymerase gene, the target gene after evolution refers to a gene with T3 RNA polymerase function (or further including T7 RNA polymerase function), and the genetic element on CCP2 refers to the T3 promoter, which controls the expression of the downstream gIII gene, thereby supporting the propagation of the gIII-deficient phage LWSe. Thus, the T7 RNA polymerase gene is functionally linked to the T7 promoter, and the T3 RNA polymerase gene is functionally linked to the T3 promoter. Furthermore, in one embodiment of the present invention, IMP1 is driven by the T7 RNA polymerase that expresses the target gene carried by LWSe before evolution and induces mutational evolution of LWSe. IMP2 is driven by the T3 RNA polymerase that expresses the target gene carried by LWSe after evolution and induces LWSe to continue mutational evolution.

[0051] As is well known, the present invention is not limited to the above example in which the "T7 RNA polymerase gene" is used as the pre-evolved target gene, and any similar technical solution can be used. Specifically, when the target gene is a protease gene, a cellulase gene, a fluorescent protein gene, a density-sensing gene, an antibody gene, or another similar target gene, their functions can be linked to the function of the helper plasmid using different principles.

[0052] For ease of understanding, we will briefly introduce several methods for linking target gene activities to gIII on a CCP in this invention. There are many methods for linking target gene activities to gIII, and these methods are not limited to those of the present invention. In the case of a protease gene, (1) the target protease's cleavage sequence is expressed as a linked fragment, fused with an auxiliary protein (such as the g6 protein of M13 phage) that blocks the gIII gene and gIII protein. The portion of gIII fused with the protein is blocked and inactive. The target protease evolves a specific activity and can only release active gIII by cleaving the designated cleavage sequence. (2) Using T7 polymerase as a medium, the target protease's cleavage sequence is expressed as a linked fragment, fused with T7 polymerase and T7 lysozyme, and a new polymerase is formed. The activity of the new polymerase is blocked by T7 lysozyme. Only the evolving protease can recognize the designated cleavage sequence and excise the lysozyme portion, resulting in an active polymerase. In the case of cellulase genes, the expression of the gIII gene can be controlled by the lactose operon, which is repressed by glucose, a product of enzymatic hydrolysis of cellulose. In the case of fluorescent protein genes, the expression of the gIII gene can be promoted by a light-inducible promoter that is sensitive to the fluorescence emitted by the target fluorescent protein. In the case of density-sensing genes, the expression of the gIII gene can be controlled by a density-sensing system. In the case of antibody genes, the antibody can be expressed by fusing it with a transcription factor that controls gIII expression, and then fusing the antibody binding site with a transcriptase.

[0053] Negative screening (b) in Figure 2 illustrates a technical solution further improving the present invention. The system further includes host bacteria S2 or S3. Host bacteria S2 contains helper plasmid CCP2, mutagenesis plasmid IMP2, and helper plasmid CCP3, which support the propagation of evolved LWSe. Host bacteria S3 contains helper plasmid CCP2, mutagenesis plasmid IMP2, and helper plasmid CCP4, which support the propagation of evolved LWSe. Helper plasmid CCP2 and mutagenesis plasmid IMP2 are similar to those in positive screening (a), except that CCP1 is a high-copy plasmid and CCP2 is a low-copy plasmid. IMP1 and IMP2 are driven by the target genes before and after evolution, respectively. However, the function of the genetic elements on CCP3 and CCP4 is related to the function of the target genes before evolution, and CCP3 and CCP4 are functionally defective and therefore cannot support the propagation of the pre-evolved LWSe. In one embodiment of the present invention, the genetic element on the helper plasmids CCP3 and CCP4 is a T7 promoter, which controls the expression of the gIII-neg gene. gIII-neg is a truncated gIII gene lacking approximately 70 amino acids between aa 280 and aa 350, and therefore cannot support the propagation and evolution of LWS. When gIII-neg and gIII are co-expressed, they competitively inhibit LWS propagation. As is well known, gIII-neg is merely an example of the present invention, and different genes can be used in different phages, and different mutations can be generated in the same gene. In one embodiment of the present invention, IMP2 is driven by T3 RNA polymerase, which expresses the evolved target gene possessed by LWSe after evolution, and induces LWSe mutational evolution. In practical applications, IMP1 and IMP2 can be induced and expressed in the same manner (e.g., induced and controlled by the psp promoter), in which case the same IMP3 plasmid can be used instead of IMP1 and IMP2. The psp promoter can detect M13 phage infection of bacteria.The psp promoter is normally autorepressed, preventing the expression of genes controlled by it. However, upon infection, the psp promoter becomes activated, allowing efficient expression of genes controlled by it. Because IPM3 promoter expression is independent of the presence or absence of target genes carried by phage LWS, using IPM3 instead of IPM1 and IPM2 does not require the replacement of IMP plasmids for positive and negative screening. The IMP1, IMP2, and IMP3 plasmids used for generating mutations are collectively referred to as IMP.

[0054] In a preferred embodiment of the present invention, the mutagenesis plasmid IMP contains a mutator gene, which is at least one of the following: DNAQ926, a DNAQ gene mutant in which the amino acids at positions 12 and 14 are mutated to Ala; the deoxyadenosine methylase dam gene; the hemimethylated GATC-binding protein seqA gene; the activation-induced cytosine deaminase gene AID; the phage PBS2 uracil DNA glycosylase inhibitor gene Ugi; and the transcriptional repressor emrR. These genes can disrupt the DNA replication process and increase the mutation frequency. In fact, any gene that can increase mutation efficiency can be used as a mutator gene in the present invention.

[0055] The directed evolution method provided by the present invention involves growing and moving host bacteria S1 carrying IMP1 and CCP1 on a culture plate, contacting phage LWS as the host bacterium S1 moves and evolving with LWS until a phage LWSe carrying the evolved target gene is produced. The phage LWS is growth-defective, and the pre-evolved LWS is capable of low-level infection and proliferation due to background expression of CCP1. The function of at least a portion of the evolved target gene is associated with the function of genetic elements on CCP2 that support the proliferation of the evolved LWSe. Therefore, LWSe can carry out highly efficient infectious proliferation in S1 with CCP1. The highly efficient infectious proliferation of the phage inhibits the growth of the host bacterium, resulting in the formation of a clear infection spot in the LWSe-infected area with few bacteria and visible to the naked eye. The infection spot allows direct analysis of the effects of evolution.

[0056] The improved method can further include growing and moving host bacterium S2 or S3 on a culture plate. As host bacterium S2 moves, it comes into contact with the initially evolving phage LWSe and continues to evolve with LWSe until phage LWSeN, which carries the evolving target gene, is produced. The host bacterium S2 contains a helper plasmid CCP2 that supports the growth of evolved LWSe, a helper plasmid CCP3 that suppresses the growth of pre-evolved LWS, and a mutagenesis plasmid IMP2. The host bacterium S3 contains a helper plasmid CCP2 that supports the growth of evolved LWSe, a helper plasmid CCP4 that suppresses the growth of pre-evolved LWS, and a mutagenesis plasmid IMP2. The function of at least a portion of the evolved target gene is related to the function of the genetic elements on CCP2 that support the growth of evolved LWS. The function of the genetic elements on CCP3 and CCP4 is related to the function of the pre-evolved target gene, and the genetic elements carried by CCP3 and CCP4 are functionally defective and therefore unable to support the growth of pre-evolved LWS. This reduces the wild-type activity of the target gene on the LWSeN. Therefore, if the wild-type activity of the target gene on the LWSeN is sufficiently low, highly efficient infectious replication can be achieved by CCP2 in the S2. Highly efficient phage replication inhibits the growth of the host bacterium, resulting in the formation of a clear infection spot in the LWSeN infection area with few bacteria and visible to the naked eye. This infection spot allows direct analysis of the effects of evolution. CCP4 is a high-copy plasmid with high levels of expression of the defective genetic element. In other words, CCP4 is an enhanced version of CCP3. Phages evolving in the S2 host can be further evolved by using S3 host bacteria instead of S2 host bacteria. Similarly, the copy number of the CCP2 plasmid is lower than that of CCP1, and the low background expression of the low-copy CCP2 prevents replication of the pre-evolved LWS phage, providing stronger selective pressure for evolution.

[0057] Furthermore, as the host bacteria continue to move out, phages with low proliferation efficiency and low wild-type target gene activity can be eluted. Furthermore, target gene activity and phage proliferation activity can be demonstrated by observing the size of the evolutionary infection spot, the number of plaques, or real-time monitoring of the reporter gene.

[0058] CCP2.1 was obtained by modifying the T3 promoter controlling the gIII gene on CCP2 with a T7 promoter. Host bacterium S4 has CCP2, and host bacterium S5 has CCP2.1. The present invention compares the activity of T7 and T3 polymerases of evolved target genes on LWSeN by measuring the difference in the number of plaques formed by the evolved phage LWSeN on S4 and S5 host bacteria, respectively.

[0059] Hereinafter, the present invention will be described in detail using the example of evolving the T7 RNA polymerase gene T7 RNAP, which recognizes the T7 promoter (SEQ ID NO: 1), into the T3 polymerase gene T3 RNAP, which recognizes the T3 promoter (SEQ ID NO: 2). However, the protection of the present invention is not limited to the evolution of the T7 RNA polymerase gene. The T3 polymerase is obtained through evolution experiments and refers to a polymerase that can functionally recognize the T3 promoter, but it does not mean that the polymerase has the same gene sequence as the natural T3 RNA polymerase. The target gene contained in and evolved by phage LWS is the T7 RNA polymerase gene. Expression of the gIII gene on helper plasmid CCP1 (SEQ ID NO: 3) is controlled by a T3 promoter. Expression of the gIII gene on helper plasmid CCP2 (SEQ ID NO: 4) is controlled by a T3 promoter. Expression of the gIII gene on helper plasmid CCP2.1 (SEQ ID NO: 6) is controlled by a T7 promoter. Expression of the gIII-neg gene on helper plasmid CCP3 (SEQ ID NO: 5) is controlled by a T7 promoter. Expression of the gIII-neg gene on helper plasmid CCP4 is controlled by a T7 promoter. gIII-neg is a truncated gIII gene lacking approximately 70 amino acids between aa 280 and aa 350 of the gIII gene, and therefore cannot support the growth and evolution of LWS. When both gIII-neg and gIII are expressed, they competitively inhibit LWS growth.

[0060] In the present invention, the phage LWS and the helper plasmid CCP4 are the same as the phages SM and HP4, respectively, disclosed in the invention of application number 201610349254.3.

[0061] Expression of the mutator gene on the mutagenesis plasmid IMP1 is controlled by the T7 promoter, whereas expression of the mutator gene on the mutagenesis plasmid IMP2 differs from that on the mutagenesis plasmid IMP1 in that it is controlled by the T3 promoter. The gene sequence of the mutagenesis plasmid IMP2 is shown in SEQ ID NO:7. Mutagenesis plasmid IMP3 differs from mutagenesis plasmid IMP2 in that expression of the mutator gene on the mutagenesis plasmid IMP3 is controlled by the psp promoter. Phage M13-WT is a wild-type phage obtained by inserting a gene into M13KO7 phage purchased from NEB. Compared to phage LWS, the genomes and functions of M13-WT (NCBI ACCESSION: V00604) and T7 phage (NCBI ACCESSION: NC_001604) are intact and can independently infect and replicate in host bacteria.

[0062] Some of the phages and plasmids of the present invention were obtained by the inventor of the present invention through further improvement of some of the materials provided by David R. Liu's laboratory. Their genetic information has already been described in a specified document (Nat Chem Biol. 2014 March;10(3):216-222). Other plasmids and strains were obtained through research by the inventor of the present invention. The host bacterium of the present invention is E. coli M15, which was obtained by introducing an F plasmid into E. coli MG1655 of the E. coli K12 series, and its genotype is F'proA + B + lacI q Δ(lacZ)M15 zzf::Tn10(Tet R ) / attB::aph tetR.

[0063] The host bacterium of the present invention is not limited to E. coli M15 but can be any E. coli strain possessing the F factor. Furthermore, the E. coli M15 and LWS, CCP1, CCP2, CCP2.1, CCP3, CCP4, IMP1, IMP2, and IMP3 of the present invention (Figures 4-12) all have genetic maps and sequences that can be referenced and obtained by conventional molecular cloning methods such as PCR, enzyme digestion and ligation, and gene recombination. Molecular cloning methods such as gene recombination, PCR, enzyme digestion and ligation are well known in the art, and specific strains, plasmids, and phages can be obtained by these methods. Therefore, the host bacterium, plasmids, and phages of the present invention have reproducible characteristics, and can be obtained by those skilled in the art using conventional methods. As those skilled in the art will understand, the present invention can be fully practiced without providing bacterial species storage.

[0064] In this study, the host bacterium harboring CCP1 and IMP1 is referred to as S1; the host bacterium harboring IMP2, CCP2, and CCP3 is referred to as S2; and the host bacterium harboring IMP2, CCP2, and CCP4 is referred to as S3. In positive screening, the initial LWS undergoes continuous directed evolution in S1. The initial LWS harbors the T7 RNAP gene and undergoes low-level growth and mutational evolution due to background expression of CCP1 only in S1. The T7 RNAP gene harbored by the LWS continues to evolve toward T3 RNAP, producing an LWS mutant strain that promotes gIII expression under the control of the T3 promoter in CCP1. This mutant strain then undergoes higher levels of growth and evolution in S1, further improving the promotion activity of gIII under the control of the T3 promoter in CCP1, resulting in the mutant phage LWSe and the target gene evolved into pre-T3 RNAP (Figure 2a). This process is called positive screening.

[0065] The evolved pre-T3RNAP on LWSe has high activity toward both T7 and T3 promoters. To improve the specificity of pre-T3RNAP toward the T3 promoter, we need to evolve and screen for T3RNAPs with poor recognition and promotion toward the T7 promoter. This process is called negative screening.

[0066] Negative screening requires the use of CCP3 or CCP4. When the evolved LWSe in the positive screening infects S2 bacteria, the evolved target gene pre-T3RNAP on LWSe can promote the expression of gIII-R5, thereby suppressing its proliferation. This situation continues until LWSe evolves several new phage mutants. The target gene carried by these new phage mutants can efficiently promote the expression of the gIII gene controlled by the T3 promoter on CCP2, but does not or only poorly promote the expression of the gIII-R5 gene controlled by the T7 promoter on CCP3 or CCP4. These new phage mutants then continue to evolve within S2 or S3 bacteria, eventually evolving to obtain the phage LWSeN carrying the highly specific T3 RNA polymerase gene T3RNAP (Figure 2b). CCP4 is a high-copy plasmid that expresses defective genetic elements at high levels. In other words, CCP4 is an enhanced version of CCP3. By using S3 host bacteria instead of S2 host bacteria, phages evolved in S2 host bacteria can be transferred to S3 host bacteria for further evolution.

[0067] The feasibility of the present invention will be explained below by way of examples. It should be noted that the following examples are merely illustrative and are intended to illustrate the feasibility of the present invention, but are not intended to limit the scope of protection of the present invention. [Example]

[0068] Example 1 Before conducting the evolution experiment, make the following preparations:

[0069] 1) The host bacterium S1 carrying the CCP1 and IMP1 plasmids was placed in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol, and then incubated at 37°C and 220 rpm until the OD 600 Then, S1 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S1 can be used for evolution experiments.

[0070] 2) The host bacterium S2 carrying the CCP2, CCP3, and IMP2 plasmids was placed in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, 50 μg / ml carbenicillin, and 25 μg / ml chloramphenicol, and then incubated at 37°C and 220 rpm until the OD 600 Then, S2 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S2 can be used for evolution experiments.

[0071] 3) The host bacterium S3 carrying the CCP2, CCP4, and IMP2 plasmids was placed in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, 50 μg / ml carbenicillin, and 25 μg / ml chloramphenicol, and then incubated at 37°C and 220 rpm until the OD 600 Then, S3 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S3 can be used for evolution experiments.

[0072] 4) The host bacterium S4 carrying CCP2 was placed in LB medium containing 50 μg / ml tetracycline and 50 μg / ml carbenicillin, and the OD was measured at 37°C and 220 rpm. 600Then, S4 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S4 is ready for use in experiments.

[0073] 5) The host bacterium S5 carrying CCP2.1 was placed in LB medium containing 50 μg / ml tetracycline and 50 μg / ml carbenicillin, and the OD was measured at 37°C and 220 rpm. 600 Then, S5 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S5 can be used for experiments.

[0074] 6) The host bacterium S6 carrying CCP1 was placed in LB medium containing 50 μg / ml tetracycline and 50 μg / ml spectinomycin, and the OD was measured at 37°C and 220 rpm. 600 Then, S6 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S6 can be used for evolution experiments.

[0075] 7) The host bacterium S7 carrying IMP3 and CCP1 was placed in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol, and then incubated at 37°C and 220 rpm until the OD 600 Then, S7 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, the host bacterium S7 is ready for use in experiments.

[0076] 8) After E. coli M15 was placed in LB medium containing 50 μg / ml of tetracycline, the OD was measured at 37°C and 220 rpm. 600 Next, E. coli M15 was diluted 100-fold and cultured again under the same culture conditions until the OD 600After two cultures, E. coli M15 can be used for experiments.

[0077] 9) After E. coli MG1655 was placed in LB medium, the OD was measured at 37°C and 220 rpm. 600 Next, E. coli MG1655 was diluted 100-fold and cultured again under the same culture conditions until the OD 600 After two cultures, E. coli MG1655 is ready for use in experiments.

[0078] Example 2 Observation method for LWS plaques in S4 or S5 host bacteria

[0079] 1) Spread one layer of 10 ml of 1.5% agarose gel on a 10 cm bacterial culture plate, and then leave it at room temperature for 20 minutes to solidify.

[0080] 2) If the concentration of phage LWS is unknown, 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 A two-fold serial gradient dilution is required.

[0081] 3) Take multiple sets of 200 μL of the S4 or S5 host bacteria prepared in "Example 1," add 10 μL of the LWS prepared in "2)" with different dilution gradients to each set, store at 55°C, and then add 4 ml of LB medium containing 0.4% bacteriological agar and carbenicillin to a final concentration of 50 μg / ml. After mixing uniformly using a vortex mixer, spread the sample onto the plate prepared in "1)" and leave it at room temperature for 1 hour to solidify the sample.

[0082] 4) Incubate overnight in a biochemical incubator at 37°C.

[0083] 5) The number of plaques formed by each gradient diluted sample is detected and counted, and the concentration of the original sample LWS is calculated.

[0084] The gIII gene of CCP2.1 carried by S5 is driven by a T7 promoter, and phage LWS carrying a T7 RNAP target gene can form plaques in S5 host bacteria. The gIII gene of CCP2 carried by S4 is driven by a T3 promoter, and LWSe with T3 RNAP activity, driven solely by the target gene, can form plaques in S4 host bacteria. The effect of LWSe evolution can be directly analyzed by comparing the changes in the number of plaques formed by LWSe in S4 and S5 host bacteria.

[0085] Example 3 Host bacteria, phage infection and translocation tests

[0086] 1) Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm bacterial culture plate. The medium contains 50 μg / ml tetracycline and 50 μg / ml carbenicillin. Allow it to solidify at room temperature for 1 hour.

[0087] 2) As shown in Figure 1a, 2 μl of the host bacteria S5 prepared in "Example 1" is inoculated onto the central surface of the plate. 1 cm away from the S5 inoculation site, three outer corners are inoculated with 10% stock concentration. 6 2 μl of LWS phage (pfu / ml) was inoculated into each well.

[0088] 3) The plate was placed in a biochemical incubator at 37°C and incubated overnight. The phenomenon shown in Figure 1b was then observed. The host bacteria migrated from the center to the edges. As they came into contact with the phages, they became infected and produced progeny phages, then continued to migrate toward the outer edge. Because the infected host bacteria grew slowly, the number of bacteria was relatively small. Because the host bacteria in the uninfected area maintained their original state, the number of bacteria was relatively large. Therefore, it was possible to observe that there were few bacteria in the infected area, and clear, V-shaped infection spots were formed. The clear infection spots indicated phage infection, and the transparency and size of the clear spots indicated the infection activity of the phage.

[0089] Example 4 SPACE Positive Screening Evolution

[0090] 1) First evolution: Prepare three 10 cm bacterial culture plates (a, b, and c). Add 10 ml of LB medium containing 0.25% bacteriological agar to each culture plate. The medium contains 50 μg / ml tetracycline and 50 μg / ml spectinomycin. Allow to solidify by leaving at room temperature for 1 hour.

[0091] Prepare four 10 cm bacterial culture plates (d, e, f, and g). Add 10 ml of LB medium containing 0.25% bacteriological agar to each culture plate. The medium contains 50 μg / ml tetracycline, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol. Allow to solidify at room temperature for 1 h.

[0092] 2) As shown in Figure 1a, 2 μl of the host bacteria S6 prepared in "Example 1" was inoculated onto the central surfaces of three plates a, b, and c. 1 cm away from the S6 inoculation site, three outer corners of plate a were inoculated with 10 μl of the stock concentration. 8 Inoculate 2 μl of LWS phage at a stock concentration of 10 pfu / ml onto the three outer corners of plate b, 1 cm away from the S6 inoculation site. 9Inoculate 2 μl of LWS phage at a stock concentration of 10 pfu / ml onto the three outer corners of plate c, 1 cm away from the inoculation site. 10 2 μl of LWS phage (pfu / ml) was inoculated into each well.

[0093] 2 μl of the host bacteria S1 prepared in "Example 1" is inoculated onto the central surfaces of four plates d, e, f, and g. 1 cm away from the S1 inoculation site, three outer corners of plate d are inoculated with 10 μl of the stock concentration. 8 Inoculate 2 μl of LWS phage at a stock concentration of 10 pfu / ml onto the three outer corners of the plate e, 1 cm away from the S1 inoculation site. 9 Inoculate 2 μl of LWS phage at a stock concentration of 10 pfu / ml onto the three outer corners of plate f, 1 cm away from the S1 inoculation site. 10 Inoculate 2 μl of LWS phage (pfu / ml) on each plate. Do not inoculate plate g with phage.

[0094] Plates d, e, and f are SAPCE evolution groups to which different amounts of initial phages were added; plates a, b, and c are control groups to which different amounts of phages were added but which did not contain the mutagenesis plasmid IMP1; and plate g is a negative control group to which no phages were added.

[0095] 3) Place the plate in a biochemical incubator at 37°C and incubate overnight.

[0096] 4) The incubation results for plates a-g are shown in 4a-g. The S6 host bacteria in plates a, b, and c do not carry the mutagenizing plasmid, and the phage cannot undergo mutational evolution. On plates a, b, and c, phage LWS grows weakly, driven only by background expression of CCP1, and is immediately shaken off by the moving host bacteria, failing to form visible infection spots. The final results are the same as those for the negative control in group g.

[0097] The host bacterium S1 in plates d, e, and f carries the mutagenizing plasmid IMP1. Therefore, initially, phage LWS is stimulated by only background expression of CCP1 and replicates and grows. However, phage LWS can induce mutational evolution of T3RNAP activity by promoting the mutagenizing gene on IMP1. Therefore, infection spots immediately form on plates d, e, and f, and the more phage initially added, the more obvious the infection spots become.

[0098] The infection spots on plate e are obvious, and no nonspecific infection spots are formed on plate b, which was added with the same amount of initial phage LWS as in plate e. Therefore, plate e was selected for experimental analysis in the following examples.

[0099] The evolved infection spot on plate e contains the evolved phage LWSe. Five-microliter samples were collected from the start point α (near the center of the plate), the midpoint β, and the end point γ (away from the center) of the infection spot on plate e, designated LWSeα, LWSeβ, and LWSeγ. The samples were diluted 100-fold with LB liquid medium and mixed uniformly for 2 minutes using a vortex mixer. The uniformly mixed samples were then filtered through a 0.22-micrometer filter. The formation of T3 plaques in S4 host bacteria, where gIII gene expression is controlled by the T3 promoter, and the formation of T7 plaques in S5 host bacteria, where gIII gene expression is controlled by the T7 promoter, were detected using the method described in "Example 2."

[0100] As shown in Figure 4h, the phage LWS before evolution possesses a wild-type T7RNAP gene and can form T7 plaques only in host bacterium S5, but is unable to form T3 plaques in host bacterium S4. As evolution progresses, phage LWS becomes phage LWSe. The target gene carried by LWSe undergoes constant mutation, resulting in the development of T3 RNAP activity, the ability to recognize the T3 promoter, and the ability to form T3 plaques in host bacterium S4. Furthermore, the further away from the center of the plate, the longer the evolution time and the more effective the evolution, resulting in the more evident T3 RNAP activity obtained by LWSe. As shown in Figure 4h, LWS~LWSeγ exhibits a stronger ability to form T3 plaques in S4. Therefore, in the examples that follow, samples are taken from the end of the infection spot away from the center of the plate at each evolution stage, diluted 100-fold, filtered through a 0.22 μm small filter, and then analyzed or evolved further.

[0101] To ensure consistency with subsequent examples, the LWSeγ sample will be referred to as LWSe1, where "1" indicates that it has been evolved once. In this invention, LWSe1 and LWSeγ represent the names of the same sample in different scenes.

[0102] Second evolution: On the same plate, inoculate the S1 host bacteria in the center of the plate, and inoculate 2 μl of filtered phage LWSe1 at each of the three corners 1 cm away from the S1 inoculation site, and perform a second evolution under the same conditions.

[0103] At the end of the second round of evolution, 5 μl of the LWSe2 phage sample was diluted 100-fold and filtered through a 0.22 μm small filter to detect the formation of T3 plaques in S4 host bacteria and T7 plaques in S5 host bacteria. As shown in Figure 5, after one additional round of evolution, LWSe2's ability to form T3 plaques in S4 host bacteria was stronger than that of LWSe1.

[0104] Example 5 SPACE negative screening evolution with S2 or S3 host bacteria

[0105] 1) Third round of evolution: Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm culture plate. The medium contains 50 μg / ml tetracycline, 50 μg / ml carbenicillin, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol. Allow to solidify at room temperature for 1 hour. The CCP3 and CCP4 plasmids carried by S2 contain not only a T7 promoter but also a theophylline-inducible riboswitch. Because the riboswitch is not strict, there is constant background expression of gIII-neg even without theophylline, suppressing the growth of phage LWSe, which carries the target gene and has wild-type T7 RNAP activity.

[0106] Inoculate 2 μl of host bacteria S2 in the center of the plate, then inoculate 2 μl of filtered phage LWSe2 at each of the three corners, 1 cm from the S2 inoculation site, and culture overnight in a 37°C incubator for a third round of evolution.

[0107] At the end of the third evolutionary infection spot, 5 μl of the LWSe3 phage sample was taken, diluted 100-fold, and filtered through a 0.22 μm small filter to detect the formation of T3 plaques on S4 host bacteria and T7 plaques on S5 host bacteria, respectively.

[0108] 2) Fourth Evolution: The filtered LWSe3 sample was subjected to a fourth evolution under the same conditions as in step 1 of this example. Similarly, at the end of the fourth evolution infection spot, 5 μl of the LWSe4 phage sample was taken and diluted 100-fold, and filtered through a 0.22 μm small filter to detect the formation of T3 plaques (plaques) formed by the sample on S4 host bacteria and T7 plaques (plaques) formed by the sample on S5 host bacteria.

[0109] 3) Fifth round of evolution: Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm culture plate. The medium contains 50 μg / ml tetracycline, 50 μg / ml carbenicillin, 50 μg / ml spectinomycin, 25 μg / ml chloramphenicol, and 1 mM theophylline. Allow to solidify at room temperature for 1 hour. Adding theophylline increases the expression of gIII-negative CCP3 and CCP4, thereby increasing the pressure of evolutionary screening.

[0110] Inoculate 2 μl of host bacteria S2 in the center of the plate, then inoculate 2 μl of filtered phage LWSe4 at each of the three corners, 1 cm away from the S2 inoculation site, and culture overnight in a 37°C incubator. Perform the fifth round of evolution.

[0111] At the end of the fifth evolutionary infection spot, 5 μl of the LWSe5 phage sample was taken, diluted 100-fold, and filtered through a 0.22 μm small filter to detect the formation of T3 plaques on S4 host bacteria and T7 plaques on S5 host bacteria, respectively.

[0112] 4) Sixth Evolution: Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm culture plate. The medium contains 50 μg / ml tetracycline, 50 μg / ml carbenicillin, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol. Allow to solidify at room temperature for 1 hour.

[0113] Inoculate 2 μl of the host bacteria S3 in the center of the plate, then inoculate 2 μl of the filtered phage LWSe5 at each of the three corners, 1 cm from the S3 inoculation site, and culture overnight in a 37°C incubator for a sixth round of evolution.

[0114] At the end of the sixth evolutionary infection spot, 5 μl of the LWSe6 phage sample was taken, diluted 100 times, and filtered through a 0.22 μm small filter to detect the formation of T3 plaques in S4 host bacteria and T7 plaques in S5 host bacteria.

[0115] 5) Seventh Evolution: Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm culture plate. The medium contains 50 μg / ml tetracycline, 50 μg / ml carbenicillin, 50 μg / ml spectinomycin, 25 μg / ml chloramphenicol, and 1 mM theophylline. Allow to solidify at room temperature for 1 hour.

[0116] Inoculate 2 μl of host bacteria S3 in the center of the plate, then inoculate 2 μl of filtered phage LWSe6 at each of the three corners, 1 cm away from the S3 inoculation site, and culture overnight in a 37°C incubator. Perform the seventh round of evolution.

[0117] At the end of the seventh evolutionary infection spot, 5 μl of the LWSe7 phage sample was taken and diluted 100 times. After filtering through a 0.22 μm small filter, the formation of T3 plaques on S4 host bacteria and T7 plaques on S5 host bacteria was detected by the sample.

[0118] The effects of the seventh round of evolution are shown in Figure 5. Part a of Figure 5 shows the copy numbers of different CCPs. The copy number affects the gene expression level and therefore the selective pressure during the evolutionary process. Part b of Figure 5 shows the effect of each evolutionary step on the plate. The color of the evolutionary pressure line in part b, which changes from light to dark, indicates that the evolutionary selective pressure changes from weak to strong under different evolutionary conditions. Part c of Figure 5 shows the plaque formation effect of each evolutionary step on S4 and S5 host bacteria. As shown in the figure, as the number of evolutionary rounds increases, the selective pressure during the evolutionary process becomes stronger, and the effect of evolution becomes more apparent. The LWS phage, which harbors the wild-type T7 RNAP gene, can form T7 plaques in S5 host bacteria, where gIII gene expression is controlled solely by the T7 promoter, but cannot form T3 plaques in S4 host bacteria, where gIII gene expression is controlled by the T3 promoter. As evolution progresses, the ability of phages LWSe1 through LWSe7 to form T7 plaques on S5 host bacteria generally weakens, but their ability to form T3 plaques on S4 host bacteria generally strengthens.

[0119] Example 6 Sequencing analysis of SPACE products

[0120] For the phages LWSe1 and LWSe2, the products of the first two rounds of evolution, several clones were purified and the target genes were sequenced. As shown in Figure 5d, the target genes carried by the phages all had corresponding mutations in amino acids 1-310 of the N-terminus. This region has been reported to be involved in promoter recognition by T7 RNAP. David R. Liu has reported that the E222K mutation affects the specificity of T7 RNAP.

[0121] Comparing the ability of the phages before and after evolution to form plaques in S5 and S4 host bacteria, as well as the sequencing results, indicates that the change in the activity of the target gene carried by LWSe phage from T7RNAP to T3RNAP is caused by mutations during SAPCE evolution.

[0122] Experimental Example 1 IMP3-guided SPACE evolution test

[0123] 1) Prepare three 10 cm bacterial culture plates a, b, and c. Add 10 ml of LB medium containing 0.25% bacteriological agar to each culture plate. The medium contains 50 μg / ml tetracycline, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol. Allow to solidify by leaving at room temperature for 1 hour.

[0124] 2) As shown in Figure 1a, 2 µl of the host bacteria S7 prepared in Example 1 was inoculated onto the central surface of each of plates a and c. Plate a was inoculated with 10 µl of the host bacteria S7 at a stock concentration of 10 µL at the three outer corners 1 cm away from the inoculation site of the host bacteria S7. 9 2 μl of LWS phage (pfu / ml) was inoculated. No phage was inoculated on plate c, the negative set.

[0125] 2 μl of the host bacteria S1 prepared in Example 1 is inoculated onto the central surface of plate b. 1 cm away from the inoculation site of the host bacteria S1, three outer corners of plate b are inoculated with 10 92 μl of LWS phage (pfu / ml) was inoculated into each well.

[0126] 3) Place the plate in a biochemical incubator at 37°C and incubate overnight.

[0127] 4) The evolutionary effects of plates a-c are shown in Figures 6a-6c. Similar infection spots were formed on the host bacteria S7 carrying IPM3 and the host bacteria S1 carrying IMP1. At the end of the evolutionary infection spot on the host bacteria S7, 5 μl of the evolved phage sample LWSeP1 was taken and diluted 100-fold and then filtered through a 0.22 μm small filter. The formation of T3 plaques on the S4 host bacteria and T7 plaques on the S5 host bacteria was then monitored.

[0128] At the end of the infection spot of the S1 host bacteria set, 5 μl of the evolved phage sample LWSeT1 was taken and diluted 100-fold, then filtered through a 0.22 μm small filter. The formation of T3 plaques on the S4 host bacteria and T7 plaques on the S5 host bacteria was then detected.

[0129] As shown in Figure 6d, the numbers of T3 and T7 plaques in LWSeT1 and LWSeP1 are similar, indicating that the effects of IMP3 and IMP1, which are controlled by the psp operon, on promoting mutation evolution are similar.

[0130] Thus, by directly using IMP3 instead of IMP1 and IMP2, we can decouple IMP expression from target gene activity, allowing us to use the same IMP3 for both positive and negative screening, regardless of the evolutionary synthesis of the target gene.

[0131] Experimental Example 2 Infectious transfer test of virulent phages

[0132] 1) The above example is carried out using the chronically infecting M13 phage. However, the present invention can also be used to carry out evolution experiments using other phages. In this example, the virulent phage T7 phage is used as an example.

[0133] 2) Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm bacterial culture plate and allow it to solidify by leaving it at room temperature for 1 hour.

[0134] 3) As shown in Figure 1a, 2 μl of the host bacteria E. coli MG1655 prepared in "Example 1" is inoculated onto the central surface of the plate. 1 cm away from the E. coli MG1655 inoculation site, three outer corners are inoculated with 10% stock concentration. 4 , 10 5 , 10 6 Inoculate 2 μl of T7 phage (pfu / ml) into each well. In the control group, use LB medium instead of phage.

[0135] 4) Place the plate in a biochemical incubator at 37°C and incubate overnight. After that, the phenomenon shown in Figure 7b can be observed. The host bacteria move from the center to the edge, and as they move, they come into contact with the phages, become infected, produce progeny phages, and continue to move to the outer edge. T7 phage is a strong phage, and any host bacteria it infects will decompose and die. Therefore, a V-shaped transparent infection spot will form on the plate. Since there are no phages in the control group 16a, no V-shaped infection spot will form, and the bacteria will be evenly distributed in a circle. The inoculation amount is 10 6 In the set of pfu / ml, samples were taken at point α of the infected area, point β of the uninfected area, and point γ of the crossover area in Figure 7b, and the number of plaques was counted using E. coli MG1655 according to "Example 2." 9T7 phage was detected at pfu / ml levels. T7 phage infects the host bacteria while co-moving with them on the plate, and infection can be indicated by visible infected spots. The examples of the present invention performed with M13 phage can also be performed with T7 phage.

[0136] In the above examples, the present invention has been described by taking the example of evolving the T7 RNA polymerase gene T7RNAP, which recognizes the T7 promoter, into the T3 RNA polymerase gene T3RNAP, which recognizes the T3 promoter. However, in other examples, other target genes (such as protease genes, cellulase genes, fluorescent protein genes, and density-sensing genes) can be used in place of the T7RNAP gene in the LWS, and the expression regulation and post-expression modification methods for the gIII and gIII-R5 genes in the CCP1, CCP2, CCP3, and CCP4 plasmids can be adjusted accordingly to link the expression of gIII and gIII-R5 with the biological activity of the new target gene being evolved on the LWS. This allows the system to perform directed evolution for new target genes.

[0137] Although the present invention has been described with reference to specific examples, various changes and modifications can be made without departing from the spirit and scope of the present invention, and it goes without saying that these modifications and modifications are also included in the present invention.

Claims

1. 1. A method for visual continuous spatially directed evolution, comprising: i) inoculating a host in a first region of a solid culture space; ii) inoculating a host with a corresponding parasite in a second portion of the solid culture space, wherein a target gene to be evolved is present in the parasite, and the host itself contains a mutagenesis plasmid that assists in the evolution of the target gene and a helper plasmid that supports the growth of the parasite, and the target gene is linked to the growth and movement of the host via the helper plasmid in the host; iii) growing and moving the host in a solid culture space and infecting it with the parasite when it comes into contact with the parasite, wherein the target gene evolves as the host grows and moves in the solid culture space, and the proliferation of the parasite and the infection of the host by the parasite affect the growth and movement of the host, resulting in the formation of an infection spot visible to the naked eye; iv) screening to obtain evolved products depending on the different spatial distribution patterns formed in the solid culture space by the growth and movement of the host, wherein the evolved products are evolved target genes that enable the helper plasmid to support higher levels of growth of the parasite; Including, The method, wherein the parasite is a phage and the host is a bacterium.

2. The host is the natural host bacterium of a non-defective strain of said phage, a strain obtained by genetic recombination with the natural host bacterium of a non-defective strain of said phage, or 2. The method of claim 1, wherein the non-native host bacterium acquires susceptibility to the phage only after genetic recombination.

3. 2. The method of claim 1, wherein the host is selected from the group consisting of Escherichia coli, Pasteurella, Shigella, Pseudomonas, Xanthomonas, Salmonella, and Staphylococcus aureus.

4. The method of claim 3, wherein the host is Escherichia coli carrying the F factor.

5. The method of claim 1 , wherein the phage is a temperate phage, a virulent phage, or a chronically infecting phage.

6. 2. The method of claim 1, wherein the phage is selected from the group consisting of filamentous phage, T4 phage, T7 phage, λ phage, P1 phage, P2 phage, P22 phage, φX174 phage, and SP6 phage.

7. The method of claim 6, wherein the filamentous phage is an M13 filamentous phage or an f1 filamentous phage.

8. 2. The method of claim 1, wherein the target genes are a combination of one or more protein-coding and non-coding genes.

9. 9. The method of claim 8, wherein the target gene is selected from the group consisting of a T7 RNA polymerase gene, a protease gene, a cellulase gene, a fluorescent protein gene, and a density-sensing gene.

10. 2. The method of claim 1, wherein expression of the mutagenized plasmid is driven or induced by a gene in the pre-evolved phage or the evolved phage.

11. The method of claim 10, wherein the gene in the pre-evolved phage or the evolved phage comprises at least one selected from the group consisting of a pre-evolved target gene, an evolved target gene, a phage gene, or an introduced foreign gene.

12. The method of claim 10, wherein the mutagenizing plasmid contains a mutagenizing gene, and the mutagenizing gene includes at least one selected from the group consisting of the DNAQ926 gene, a DNAQ gene mutant in which the amino acids at positions 12 and 14 are mutated to Ala, the deoxyadenosine methylase dam gene, the hemimethylated GATC binding protein seqA gene, the activation-induced cytosine deaminase gene AID, the uracil DNA glycosylase inhibitor gene Ugi of phage PBS2, and the transcriptional repressor emrR.

13. The solid culture space is a two-dimensional planar culture structure or a three-dimensional space culture structure; The vertical movement and evolution of the solid culture space is maintained continuously by a casting solid culture system that is periodically formed.

10. The method of claim 1, wherein the directed evolution is performed by using multiple solid culture spaces or different locations of the solid culture space.

14. 2. The method of claim 1, wherein the gIII gene of the phage, which is necessary for packaging and infection of the host, is excised and the helper plasmid comprises the sequence shown in SEQ ID NO:3 or SEQ ID NO:

4.

15. 15. The method of claim 14, wherein the helper plasmid further comprises a second helper plasmid, the second helper plasmid comprising the sequence set forth in SEQ ID NO:

5.

16. 16. The method of any one of claims 1 to 15, wherein the directed evolution is performed by alternating between different hosts, and the latter hosts contain genetic elements that support the propagation of phages, the genetic elements including a helper plasmid that supports the propagation of evolved phages and a helper plasmid that suppresses the propagation of pre-evolved phages.

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