Visual continuous space directed evolution method

Abstract

The invention relates to the field of directed evolution screening, in particular to a visual continuous space directed evolution method, a host grows and moves in a solid culture space, the host carries an external target gene to be evolved, and the host comprises a gene element for assisting the evolvement of the target gene. The target gene is associated with the growth movement of the host; in the host growth movement process, different spatial distribution forms are formed in the solid culture space for screening to obtain an evolutionary product. The method is directly carried out in a solid culture space, different spatial distribution pattern images visible to naked eyes are locally formed, then evolution products are selected, liquid fed-batch culture equipment is not needed, meanwhile, the evolution effect is observed by naked eyes through infection spots formed in evolution, real-time monitoring equipment is not needed, operation is easy, and the flux is high.

Description

[0001] This application is a divisional application of Chinese patent application No. 201711446362.3 (filed on December 27, 2017, entitled "A Visual Continuous Spatial Directed Evolution Method"). Technical Field

[0002] This invention relates to the field of directed evolution screening, and more specifically, to a visual, continuous spatial directed evolution method. Background Technology

[0003] Directed evolution (also known as laboratory evolution) is a powerful technique that can manipulate the biological evolutionary process to produce biomolecules with specific functions. These biomolecules have been widely applied in industrial production, bioengineering, pharmaceutical development, and many other fields. David R. Liu's lab at Harvard University has developed a continuous evolution system based on bacteriophage growth (phage-assisted continuous evolution, PACE). It mainly consists of three modules: LWS, CCP, and IMP. The LWS module contains the gIII gene, necessary for packaging and infecting the host bacterium in the M13 gene of the bacteriophage, which is removed and replaced with the gene of the target biomolecule to be evolved. Without gIII, the bacteriophage cannot infect the host and produce progeny bacteriophages. The IMP module uses arabinose as an inducer to induce the expression of DNAQ926, dam, and seqA, preventing the removal of incorrect bases introduced by the polymerase during DNA replication and increasing the mutation rate. The IMP induced by arabinose increases the mutation rate of the bacteriophage by several hundred times. The CCP module contains the gIII gene required for the bacteriophage to infect and reproduce in the host cell. By binding the expression of gIII on the CCP to the biological activity of the target gene to be evolved on the LWS (for example, to evolve an RNA polymerase in a specific direction, a promoter consistent with the evolution direction is used to control the expression of gIII), it determines whether each mutant LWS can produce progeny bacteriophages with infectious activity.

[0004] In the evolutionary pool, when a bacteriophage carrying the wild-type target gene to be evolved infects a host cell, it injects its wild-type LWS genetic material into the host bacteria, utilizing the host bacteria's replication system to replicate its genetic material. Simultaneously, under the induction of arabinose, DNAQ926, dam, and seqA on the IMP in the host cell are expressed, leading to mutations in the LWS. If the mutation acquired by the LWS (i.e., the target gene mutation on the LWS) can initiate the expression of the gIII protein, then infectious progeny bacteriophages can be produced. These newly generated progeny bacteriophage mutant strains, LWSe, are secreted outside the host bacteria to infect new host bacteria, initiating the next round of replication and proliferation. Thus, those LWSe mutant strains that can initiate gIII expression can continuously proliferate, increasing their numbers, while wild-type LWS and mutant LWS that cannot initiate gIII expression cannot secrete progeny bacteriophages for proliferation, and their numbers do not increase. If new host bacterial cultures are continuously added to the evolution pool at a certain rate and old cultures are removed, wild-type LWS and its mutant strains that cannot initiate gIII expression or have low expression ability will be quickly washed out, while LWSe mutant strains that can efficiently initiate gIII expression will eventually be retained.

[0005] This system is quite large, requiring a complete automated fed-batch culture system with a highly sensitive real-time monitoring system for continuous fed-batch culture. Different evolutionary products are mixed and diluted together in the entire culture vessel, making direct detection and separation difficult. The system consumes a lot of reagents, the culture equipment is expensive, and the evolutionary operation is complex. Furthermore, the system can only evolve one target gene at a time, resulting in low throughput.

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

[0007] To address the problems of existing methods, such as difficulty in sample detection and separation, low throughput, high cost, and complex evolutionary operations, this invention provides a visual, continuous spatial directional evolution method. This method is performed directly on the surface of a solid culture space, such as a solid culture plate, without the need for liquid fed-batch culture equipment. At the same time, the evolutionary effect can be observed visually through the morphology and distribution of the infection spots formed during evolution, without the need for real-time monitoring equipment. This method is simple to operate, has high throughput, and can perform multiple sets of evolutionary experiments at once.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: A visual, continuous spatial directed evolution method, wherein a host grows and moves in a solid culture space, the host carries an exogenous target gene to be evolved, the host itself contains gene elements that assist the evolution of the target gene, and the target gene is associated with the growth and movement of the host; Through the host's growth process, different spatial distribution patterns are formed in the solid culture space, and evolutionary products are obtained by screening.

[0009] This invention provides a visual, continuous spatial directional evolution method (SPACE). The system includes a target gene to be evolved and a host. The host contains gene 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, resulting in different spatial distribution patterns visible to the naked eye. The evolutionary products are then selected based on these patterns.

[0010] The entire evolution and selection process unfolds in a two-dimensional plane or three-dimensional space, with different evolutionary products distributed in different regions of the plane without mixing. Furthermore, each product, based on its activity, forms a visible spatial distribution pattern locally. This pattern is continuously magnified as evolution progresses. As soon as an evolutionary product appears, an image is formed directly at its corresponding location; even in small quantities, the image is not diluted or obscured. Evolutionary products meeting specific requirements can be directly separated and selected based on the image's state. The system is simple to operate, low-cost, requires no special equipment, and allows a single person to conduct multiple evolutionary experiments at once, enabling high-throughput directed evolution of target genes.

[0011] Furthermore, the target gene is located in the host genome, plasmid, or a corresponding parasitic organism. The target gene can be inserted into the host genome through gene recombination, or it can be achieved by introducing a plasmid into the host or by allowing a parasitic organism to invade the host, thus ensuring that the host contains the target gene to be evolved.

[0012] Furthermore, the parasitic organism includes any one of bacteriophages, algaeophages, animal and plant viruses, fungal viruses, mycoplasma, chlamydia, and bacteria.

[0013] Furthermore, the parasitic organism is a bacteriophage; The host is any one of the following: The natural host bacteria of the non-deficient strain of the bacteriophage; The strain obtained by genetically modifying the natural host bacteria of the non-deficient strain of the bacteriophage; Non-natural host bacteria that acquire susceptibility through genetic modification.

[0014] Furthermore, the hosts include Escherichia coli, Pasteurella, Shigella, Pseudomonas, Xanthomonas, Salmonella, Staphylococcus aureus, and genetically modified strains that alter their susceptibility.

[0015] Preferably, the host is Escherichia coli carrying factor F.

[0016] Furthermore, the bacteriophage is a temperate bacteriophage, a virulent bacteriophage, or a chronically infectious bacteriophage.

[0017] Furthermore, the bacteriophages include filamentous bacteriophages, T4 bacteriophages, T7 bacteriophages, λ bacteriophages, P1 bacteriophages, P2 bacteriophages, and P22 bacteriophages. X174 phage, SP6 phage.

[0018] Preferably, the filamentous phage includes M13 filamentous phage and f1 filamentous phage.

[0019] In a preferred embodiment of the present invention, the bacteriophage is M13 bacteriophage, wherein the gIII gene required for packaging and infecting the host bacteria is removed; correspondingly, the gene element that assists in the evolution of the target gene, such as the helper plasmid, contains the aforementioned gIII gene. The bacteriophage can normally invade the host bacteria and replicate its DNA. However, without the presence of the relevant helper plasmid, it cannot package progeny with infectious activity.

[0020] Furthermore, the target gene is a combination of one or more protein-coding genes and non-coding genes.

[0021] Furthermore, the target gene is selected from one or more of the following: T7 RNA polymerase gene, protease gene, cellulase gene, fluorescent protein gene, and density sensing gene.

[0022] Furthermore, the gene element that assists in the evolution of the target gene is a mutation-inducing plasmid, and the mutation-inducing plasmid is initiated or induced to express by the target gene before and after evolution, respectively.

[0023] Preferably, the induced mutation plasmid contains a mutagenic gene selected from at least one of the following: DNAQ926 gene (a DNAQ gene mutant with amino acids mutated to Ala at positions 12 and 14), deoxyadenosine methyltransferase dam gene, hemimethylated GATC-binding protein seqA gene, activation-induced cytosine deaminase gene AID, uracil DNA glycosyltransferase inhibitor gene Ugi from phage PBS2, and transcriptional repressor emrR.

[0024] Mutagenic genes can increase the mutation rate of genetic information during replication and transcription. The induced mutation plasmid (IMP) can be expressed using the same method (e.g., through the PSP promoter). In different embodiments, if multiple induced mutation plasmids (IMPs) exist, such as IMP1, IMP2, and IMP3, these plasmids can be the same; alternatively, expression can be directly initiated by the target gene before and after evolution. In this case, IMP1 and IMP2 represent different IMPs.

[0025] Furthermore, the solid culture space includes a two-dimensional planar culture structure and a three-dimensional spatial culture structure.

[0026] Furthermore, the vertical movement and evolution of the solid culture space is maintained by periodically assembling a solid culture system.

[0027] Furthermore, the directed evolution is high-throughput evolution.

[0028] Furthermore, the high-throughput evolution is achieved by using multiple sets of solid culture spaces or at different locations within the solid culture space.

[0029] Furthermore, the target gene is associated with the host growth and movement via an auxiliary plasmid, the auxiliary plasmid containing at least a first auxiliary plasmid, the first auxiliary plasmid being auxiliary plasmid CCP1 or auxiliary plasmid CCP2, the nucleic acid sequence of auxiliary plasmid CCP1 being shown in SEQ ID NO: 3, and the nucleic acid sequence of auxiliary plasmid CCP2 being shown in SEQ ID NO: 4.

[0030] The helper plasmid CCP1 or CCP2 supports low-level replication and proliferation of the phage before evolution, and the increased activity of the target gene after evolution enables the helper plasmid CCP1 or CCP2 to support the phage to evolve to a higher level of replication and proliferation.

[0031] Furthermore, the helper plasmid also includes a second helper plasmid, which is helper plasmid CCP3 or helper plasmid CCP4; the nucleic acid sequence of helper plasmid CCP3 is shown in SEQ ID NO: 5.

[0032] The helper plasmids CCP3 and CCP4 have functional defects that prevent them from supporting the proliferation of pre-evolutionary phages.

[0033] Specifically, host bacteria S1, carrying IMP1 and CCP1, grows and motiles on a culture plate. During motile movement, host bacteria S1 comes into contact with bacteriophage LWS and evolves with LWS until it produces bacteriophage LWSe carrying the evolved target gene. Bacteriophage LWS has a proliferation defect; before evolution, LWS utilizes the basal expression of CCP1, allowing for low-level infection and proliferation. The function of the evolved target gene is at least partially related to the function of gene elements on CCP2 that support the proliferation of the evolved bacteriophage LWSe. Therefore, LWSe can efficiently infect and replicate within S1 using CCP1. This efficient infection and replication of the bacteriophage inhibits the growth of the host bacteria, resulting in a visible, transparent infection patch with fewer bacteria in the LWSe infection area. This infection patch allows for direct analysis of the evolutionary effect.

[0034] Furthermore, the host bacteria also include host bacteria S2 or S3, which grow and move on a culture plate. During its movement, host bacteria S2 comes into contact with the pre-evolved bacteriophage LWSe and evolves carrying LWSe until it produces a bacteriophage LWSeN carrying a further evolved target gene. The host bacteria S2 contains the helper plasmid CCP2, which supports the proliferation of the evolved bacteriophage LWSe or LWSeN; the helper plasmid CCP3, which inhibits the proliferation of the pre-evolutionary bacteriophage LWSe; and the mutagenic plasmid IMP2. The function of the evolved target gene is at least partially associated with the function of the gene elements on CCP2 that support the proliferation of the evolved bacteriophage LWSe or LWSeN. The functions of the gene elements on CCP3 and CCP4 are associated with the function of the pre-evolutionary target gene, while the gene elements carried by CCP3 and CCP4 have functional defects and cannot support the proliferation of the pre-evolutionary bacteriophage LWSe. This step can reduce the wild-type activity of the target gene on the bacteriophage LWSeN. Therefore, when the wild-type activity of the target gene in phage LWSeN is sufficiently low, CCP2 can be used in S2 for efficient infection and replication. This efficient phage infection and replication inhibits the growth of the host bacteria, resulting in a visible, transparent infection patch with fewer bacteria in the LWSeN infection area. This infection patch allows for direct analysis of evolutionary effects. The host bacteria S3 contain the helper plasmid CCP2, which supports the proliferation of the evolved LWSe or LWSeN phage; the helper plasmid CCP4, which inhibits the proliferation of the pre-evolutionary LWSe phage; and the mutation-inducing plasmid IMP2. CCP4 is a high-copy plasmid with a higher level of expression of defective gene elements. Therefore, CCP4 is an enhanced version of CCP3. S3 bacteria are used to replace S2 bacteria for further evolution of LWSe phage. Similarly, the CCP2 plasmid has a lower copy number than CCP1, and the low-copy CCP2 has a lower background expression, making it unable to support the replication of the pre-evolutionary LWSe phage, thus providing stronger evolutionary selection pressure.

[0035] Furthermore, the method also includes: when the host bacteria move to the edge of the plate, transferring the host bacteria and the evolved bacteriophage to the next plate to continue evolution.

[0036] Furthermore, the directed evolution is carried out by succession of different hosts, and the gene elements supporting phage proliferation in the latter host include helper plasmids that support the proliferation of the evolved phage and helper plasmids that inhibit the proliferation of the pre-evolutionary phage.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention provides a visual continuous spatial directional evolution method with spatial attributes. The entire evolution process unfolds in a two-dimensional planar space or a three-dimensional solid space. The space is both a support platform and a selection pressure for evolution. As long as a small number of evolution products appear, spatial distribution patterns such as plaques can be directly formed at the corresponding positions. The activity of the target gene and the proliferation activity of the phage can be displayed by real-time monitoring of the size of the infection plaques formed during evolution, the plaque count, or the reporter gene. In this way, evolution products can be directly identified and separated without liquid fed-batch culture equipment or real-time monitoring equipment.

[0038] (2) The present invention provides a visual continuous spatial directional evolution method in which evolution results appearing at different spatial locations are fixed at that location and are not diluted or mixed by other components, and can be directly separated. The signals of evolution results appearing at different spatial locations are amplified along the direction of evolution, the evolution effect is enhanced, and higher detection sensitivity is provided.

[0039] (3) The present invention provides a visual continuous spatial directed evolution method, which has simple evolution operation, high throughput, and can perform multiple sets of evolution experiments at one time. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0041] Figure 1 This is a schematic diagram illustrating the movement of host bacteria and the effect of infection spots in an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the principle of continuous spatial directed evolution in an embodiment of the present invention; Figure 3 This is a schematic diagram of a continuous spatial directed evolution model in an embodiment of the present invention; Figure 4 This is a graph showing the evolutionary results of the first round of SPACE positive selection in Embodiment 4 of the present invention; Figure 5 The diagram shows the results of multi-round SPACE evolution in embodiments 5 and 6 of this invention; Figure 6 This is a graph showing the evolutionary effect of IMP3-induced SPACE in Experiment Example 1 of this invention, as well as a bar graph showing the number of different phages. Figure 7 The image shows the infection and movement of T7 phage in Experiment Example 2 of this invention, along with a bar chart showing the number of phages. Detailed Implementation

[0042] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0043] This invention provides a visual, continuous spatial directed evolution method. When bacteriophages and host bacteria are mixed and spread on a plate, visible plaques are formed due to the growth differences between host bacteria infected with M13 bacteriophage and uninfected host bacteria. Considering the issues of specificity, sensitivity, and operability associated with reporter genes, this invention does not employ reporter genes but directly uses the plaques formed during the infection process as an indicator signal for space, thereby making space visible.

[0044] Similar to the plaque formation assay, host bacteria are inoculated in the center of the culture plate, and wild-type bacteriophages (e.g., [missing information]) are inoculated in a triangular pattern around the host bacteria. Figure 1 (a) At this point, the host bacteria grow on the plate while simultaneously moving towards the edge. The moving host bacteria come into contact with the bacteriophage, become infected, and produce progeny bacteriophages, while continuing to move outwards. Because the infected host bacteria grow more slowly, while the host bacteria in the uninfected area remain in their original state, a V-shaped infection patch with fewer bacteria can be seen in the infected area. Figure 1 (b) The presence of this transparent plaque indicates the presence of bacteriophage infection; the transparency and size of the plaque represent the infectivity of the bacteriophage.

[0045] Inspired by the inventors' earlier research on spatial evolution systems, the core design principles of SPACE are as follows: Figure 2 , Figure 3As shown: When the LWS phage carrying the target gene infects a host bacterium containing the mutagenic plasmid IMP and the helper plasmid CCP, the phage genome replicates and mutates. If the mutant progeny LWS can activate the expression of the gIII gene on CCP, then an infectious progeny phage LWSe is produced, initiating the next round of infection evolution; otherwise, an infectivity-deficient progeny phage is produced. As evolution progresses, the number of LWSe increases, while the number of wild-type LWS phages and infectious-deficient progeny phages does not increase or increases very little. In the figure, the GOI (gene of interest) on LWS represents the target gene to be evolved.

[0046] Host bacteria containing CCP and IMP were inoculated in the center of a culture plate, and bacteriophage LWS were inoculated triangularly around the host bacteria. The host bacteria then grew and divided on the plate, simultaneously using their flagella to move towards the edge and come into contact with the pre-inoculated bacteriophage LWS at specific locations. The bacteriophage LWS invaded the host bacteria and were carried forward by the host bacteria. Initially, the bacteriophage LWS could not initiate gIII expression on CCP and could only utilize the basal expression of CCP for low-level proliferation. At this stage, the bacteriophage proliferation level was very low, and its impact on the host bacteria was minimal. Infected and uninfected host bacteria grew and divided at almost the same rate, with no morphological difference. However, because the low-level proliferation rate of the bacteriophage LWS could not keep up with the growth and movement rate of the host bacteria, these inefficient bacteriophage LWS were quickly left behind by the moving host bacteria.

[0047] During its proliferation, bacteriophage LWS simultaneously undergoes mutational evolution using IMPs until it evolves into a bacteriophage LWSe capable of efficiently initiating CCP expression. At this point, the LWSe directly initiates CCP expression, enabling it to proliferate rapidly and continuously produce progeny bacteriophages to infect moving bacteria. The efficient proliferation of LWSe disrupts the growth of host bacteria, slowing their growth. Therefore, after the evolution of LWSe, a transparent region appears at the corresponding location, indicating slower host bacterial growth and fewer bacteria. As evolution continues and the bacteria infected by LWSe continue to move, this transparent region expands in the direction of movement, eventually forming a visible V-shaped infection patch on the plate. The closer to the outer edge of the infection patch, the longer the evolutionary time and the more pronounced the evolutionary effect. We can directly obtain the desired evolved LWSe bacteriophage from the infection patch.

[0048] like Figure 2 , 3As shown, one embodiment of the phage-assisted continuous spatial directed evolution system of the present invention includes two parts: positive selection (a) and negative selection (b). It should be noted that, from the perspective of the implementation of the present invention, only the positive selection (a) part is needed to achieve the goal of evolving the target gene into the evolved target gene. Therefore, the negative selection (b) part can be regarded as a further improved technical solution of the directed evolution system of the present invention. In fact, the positive selection (a) part achieves a qualitative change from the "target gene to be evolved" to the "evolved target gene," while the negative selection (b) part achieves a quantitative change by further enhancing the function of the "evolved target gene."

[0049] In this invention, the term "target gene to be evolved" (GOI) can be equated with "pre-evolutionary target gene," that is, a target gene that has not yet undergone evolutionary mutations through the system and method of this invention, and in some cases can be called a "wild-type gene." The term "evolved target gene" is relative to the "target gene to be evolved," possessing a new evolved function; however, the "evolved target gene" may still retain its original function. Of course, the original function may also have completely disappeared. In short, in this invention, as long as the target gene possesses a new evolved function, it can be called an "evolved target gene," regardless of whether the original function still exists.

[0050] It should be noted that the symbols used in this invention, such as LWS, CCP, IMP, and S, are merely exemplary symbols. The objects represented by these symbols can be converted into other expressions. For example, bacteriophage LWS can be called a pre-evolutionary bacteriophage; bacteriophage LWSe can be called an evolved bacteriophage, and is also used as a general term for all evolved bacteriophages; bacteriophage LWSeN can be called a further evolved bacteriophage, where the number N (e.g., 1, 2, 3, or other combinations of letters and numbers) indicates the number of generations of subculture or the number of evolutions, or evolution under different conditions; host bacterium S1 can be called the first host bacterium; host bacterium S2 can be called the second host bacterium; host bacterium S3 can be called the third host bacterium; helper plasmid CCP1 can be called the first helper plasmid; helper plasmid CCP2 can be called the second helper plasmid; helper plasmid CCP3 can be called the third helper plasmid; helper plasmid CCP4 can be called the fourth helper plasmid; mutagenic plasmid IMP1 can be called the first mutagenic plasmid; mutagenic plasmid IMP2 can be called the second mutagenic plasmid; and mutagenic plasmid IMP3 can be called the third mutagenic plasmid. The plasmids CCP1 and CCP2, which support the proliferation of the evolved bacteriophage LWSe, are collectively referred to as positive screen CCPs. Similarly, the plasmids CCP3 and CCP4, which inhibit the proliferation of the pre-evolutionary bacteriophage LWS, are collectively referred to as negative screen CCPs. The plasmids IMP1, IMP2, and IMP3, which are used to generate mutations, are collectively referred to as IMPs.

[0051] Furthermore, the host bacteria in this invention, in addition to the aforementioned host bacteria S1, S2, and S3, may also include other host bacteria. It should also be noted that in this invention, host bacteria S1, S2, and S3 do not represent different "species," but rather different helper plasmids or mutagenic plasmids carried by the strains. In this invention, host bacteria S1, S2, and S3 can be obtained by introducing different plasmids into the same species, such as *Escherichia coli* carrying the F factor.

[0052] like Figure 2 , 3 As shown, one embodiment of the phage-assisted continuous spatial directed evolution system of the present invention includes a phage LWS carrying a target gene to be evolved, wherein the phage LWS has a proliferation defect. A "proliferation defect" generally refers to a deficiency in certain essential functions during the phage's life cycle, such as defects in packaging and / or infection of host bacteria, which may be caused by mutations in related genes. In different phages, the genes responsible for essential functions such as packaging and / or infection of host bacteria differ. For example, in one embodiment of the present invention, the phage LWS is M13 phage, in which the gIII gene required for packaging and infection of host bacteria is removed, resulting in the inability to properly package and infect host bacteria. Those skilled in the art should understand that any similar phage can serve as the phage LWS in the present invention, and is not limited to M13 phage.

[0053] Figure 2 , 3In the directed evolution system shown, the host bacterium S1 contains the helper plasmid CCP1, which supports the proliferation of evolved LWSe, and the mutagenic plasmid IMP1. Pre-evolutionary LWSe utilizes the basal expression of CCP1 to achieve low-level infection and proliferation. The support of the helper plasmids CCP1 and CCP2 for the proliferation of evolved LWSe is achieved by the function of the evolved target gene being at least partially associated with the function of the gene elements on CCP1 and CCP2 that support the proliferation of evolved LWSe. "At least partially" means that the function of the evolved target gene, in addition to being partially associated with the function of the gene elements for the proliferation of evolved LWSe, may also be partially associated with the function of the gene elements for the proliferation of pre-evolutionary LWSe. For example, in one embodiment of the invention, the pre-evolutionary target gene is the T7 RNA polymerase gene, the evolved target gene is a gene with T3 RNA polymerase function (including T7 RNA polymerase function), and the gene element on CCP2 is the T3 promoter, which controls the expression of the downstream gIII gene to assist the proliferation of gIII-deficient phage LWSe. In this way, the T7 RNA polymerase gene is functionally associated with the T7 promoter, while the T3 RNA polymerase gene is functionally associated with the T3 promoter. Furthermore, in one embodiment of the present invention, IMP1 is initiated by the T7 RNA polymerase expressed by the target gene carried by the pre-evolutionary LWS and induces LWS mutational evolution. IMP2 is initiated by the T3 RNA polymerase expressed by the target gene carried by the post-evolutionary LWSe and induces LWSe to continue its mutational evolution.

[0054] Those skilled in the art should understand that this invention is not limited to the above-described embodiment using the "T7 RNA polymerase gene" as the pre-evolutionary target gene, but includes any similar technical solutions. Specifically, for different target genes, such as protease genes, cellulase genes, fluorescent protein genes, density sensing genes, and antibody genes, their functions can be associated with the functions of helper plasmids through different principles.

[0055] For ease of understanding, this invention briefly introduces methods for associating the activity of several other target genes with gIII on CCP. It should be noted that there are many methods for associating the activity of target genes with gIII, and it is not limited to these methods. For protease genes: (1) Using the degradation sequence of the target protease as a linker, the gIII gene is fused with an accessory protein (such as the g6 protein of M13 phage) that can block the binding of gIII protein. At this time, the gIII part of the fusion protein is blocked and has no activity. Only when the target protease evolves specific activity and degrades the specified degradation sequence can active gIII be released. (2) Using T7 polymerase as an intermediary, the degradation sequence of the target protease is used as a linker to fuse T7 polymerase with T7 lysozyme to form a new polymerase. The activity of this new polymerase is blocked by T7 lysozyme. Only when the evolved protease can recognize the set degradation sequence and remove the lysozyme part can the obtained polymerase be active. For cellulase genes: The expression of gIII gene can be controlled by using the lactose operon, which is inhibited by glucose, a product of cellulose enzymatic hydrolysis. For fluorescent protein genes: gIII gene expression can be initiated using a photoinducible promoter sensitive to the fluorescence emitted by the target fluorescent protein. For density-sensing genes: gIII gene expression can be controlled using a density-sensing system. For antibody genes: antibodies can be fused with transcription factors controlling gIII expression, while antibody binding sites can be fused with transcriptases.

[0056] Figure 2The negative selection (b) section illustrates a further improved technical solution of the present invention. The system also includes a host bacterium S2 or S3, wherein the host bacterium S2 contains an auxiliary plasmid CCP2 supporting the proliferation of post-evolutionary LWSe, a mutation-inducing plasmid IMP2, and an auxiliary plasmid CCP3. The host bacterium S3 contains an auxiliary plasmid CCP2 supporting the proliferation of post-evolutionary LWSe, a mutation-inducing plasmid IMP2, and an auxiliary plasmid CCP4. The auxiliary plasmid CCP2 and the mutation-inducing plasmid IMP2 are similar to those in the positive selection (a) section, except that CCP1 is a high-copy plasmid and CCP2 is a low-copy plasmid; IMP1 and IMP2 are initiated by the pre-evolutionary and post-evolutionary target genes, respectively. However, the functions of the gene elements on CCP3 and CCP4 are associated with the functions of the pre-evolutionary target gene, and CCP3 and CCP4 have functional defects and cannot support the proliferation of pre-evolutionary LWSe. In one embodiment of the present invention, the gene 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 defective gIII, lacking approximately 70 amino acids between aa280 and aa350 of the gIII gene, and cannot support LWS proliferation and evolution. When gIII-neg is expressed together with gIII, it competitively inhibits LWS proliferation. Those skilled in the art should understand that gIII-neg is also exemplary; different genes can be used depending on the different bacteriophages, and different mutations can be made in the same gene. Furthermore, in one embodiment of the present invention, IMP2 is initiated by the T3 RNA polymerase expressing the evolved target gene carried by the evolved LWSe, and induces LWSe mutation evolution. In fact, IMP1 and IMP2 can be induced and expressed in the same way (e.g., induced and controlled by the psp promoter), in which case IMP1 and IMP2 are replaced by the same IMP3 plasmid. The psp promoter can sense the infection of bacteria by M13 bacteriophage. The PSP promoter is normally in a state of self-suppression, and the genes it controls are basically not expressed. However, during infection, the PSP promoter is activated, and the genes it controls are expressed efficiently. Since the expression of IPM3 is independent of whether the target gene is carried on the phage LWS, when using IPM3 instead of IPM1 and IPM2, it is not necessary to change the IMP plasmid during positive and negative selection. IMP1, IMP2, and IMP3, these plasmids used to generate mutations, are collectively referred to as IMPs.

[0057] In a preferred embodiment of the present invention, the mutated plasmid IMP contains a mutagenic gene selected from at least one of the following: the DNAQ926 gene (a DNAQ gene mutant with amino acids mutated to Ala at positions 12 and 14), the deoxyadenosine methyltransferase dam gene, the hemimethylated GATC-binding protein seqA gene, the activation-induced cytosine deaminase gene AID, the uracil DNA glycosyltransferase repressor gene Ugi from bacteriophage PBS2, and the transcriptional repressor emrR. These genes can interfere with DNA replication, leading to an increased mutation frequency. In fact, any gene capable of increasing mutation efficiency can be used as a mutagenic gene in this invention.

[0058] The directed evolution method provided by this invention includes: a host bacterium S1 carrying IMP1 and CCP1 growing and motile on a culture plate; during motile movement, the host bacterium S1 comes into contact with bacteriophage LWS and evolves carrying LWS until a bacteriophage LWSe carrying the evolved target gene is produced. The bacteriophage LWS has a proliferation defect; before evolution, LWS utilizes the basal expression of CCP1 and can evolve to a low level of infection and proliferation. The function of the evolved target gene is at least partially associated with the function of gene elements on CCP2 that support the proliferation of the evolved LWSe. Therefore, LWSe can efficiently infect and proliferate in S1 using CCP1. The efficient infection and proliferation of the bacteriophage inhibits the growth of the host bacterium, resulting in a visible, transparent infection patch with few bacteria in the LWSe infection area. The evolutionary effect can be directly analyzed through this infection patch.

[0059] As a further improvement to the above method, the method further includes: host bacteria S2 or S3 growing and motile on a culture plate, respectively. During motile movement, host bacteria S2 comes into contact with the initially evolved bacteriophage LWSe and continues to evolve with LWSe until it produces a bacteriophage LWSeN carrying a further evolved target gene. The host bacteria S2 contains an auxiliary plasmid CCP2 supporting the proliferation of the evolved LWSe, an auxiliary plasmid CCP3 inhibiting the proliferation of the pre-evolutionary LWSe, and a mutation-inducing plasmid IMP2. The host bacteria S3 contains an auxiliary plasmid CCP2 supporting the proliferation of the evolved LWSe, an auxiliary plasmid CCP4 inhibiting the proliferation of the pre-evolutionary LWSe, and a mutation-inducing plasmid IMP2. The function of the evolved target gene is at least partially associated with the function of the gene elements on CCP2 that support the proliferation of the evolved LWSe. The functions of the gene elements on CCP3 and CCP4 are associated with the function of the pre-evolutionary target gene, while the gene elements carried by CCP3 and CCP4 have functional defects and cannot support the proliferation of the pre-evolutionary LWSe. This step reduces the wild-type activity of the target gene on LWSeN. Therefore, when the wild-type activity of the target gene in LWSeN is sufficiently low, CCP2 can be used for efficient infection and replication in S2. Efficient phage infection and replication inhibits the growth of the host bacteria, resulting in a visible, transparent infection patch with fewer bacteria in the LWSeN infection area. This infection patch allows for direct analysis of evolutionary effects. CCP4 is a high-copy plasmid with a higher level of expression of defective gene elements. Therefore, CCP4 is an enhanced version of CCP3. The S3 host bacteria are used to replace the S2 host bacteria, further evolving the phage that evolved in the S2 host bacteria. Similarly, the CCP2 plasmid has a lower copy number than CCP1, and the low-copy CCP2 has lower background expression, which cannot support the replication of the pre-evolutionary LWS phage, thus providing stronger evolutionary selection pressure.

[0060] Furthermore, the host bacteria can wash away phages carrying wild-type target gene activity with lower proliferation efficiency and lower content during their continuous outward movement. In addition, observing the size of evolutionary plaques, plaque counts, or real-time monitoring of reporter genes can reveal the activity of the target gene and the proliferative activity of the phages.

[0061] CCP2.1 was obtained by modifying the T3 promoter controlling the gIII gene on CCP2 to a T7 promoter. Host bacteria S4 carried CCP2, and host bacteria S5 carried CCP2.1. This invention compares the T7 and T3 polymerase activities of the evolved target gene on LWSeN by the difference in the number of plaques formed by the evolved bacteriophage LWSeN in hosts S4 and S5, respectively.

[0062] The following will use the evolution of the T7 RNA polymerase gene T7RNAP, which recognizes the T7 promoter (SEQ ID NO: 1), into the T3 polymerase gene T3RNAP, which recognizes the T3 promoter (SEQ ID NO: 2), as an example to describe the present invention in detail. However, the protection of the present invention is not limited to the evolution of the T7 RNA polymerase gene. The T3 polymerase referred to here is a polymerase obtained through evolutionary experiments that is functionally capable of recognizing the T3 promoter, but this does not mean that the polymerase has the same gene sequence as the natural T3 RNA polymerase. The target gene for evolution carried by the bacteriophage LWS is the T7 RNA polymerase gene. The gIII gene on helper plasmid CCP1 (SEQ ID NO: 3) is expressed under the control of the T3 promoter; the gIII gene on helper plasmid CCP2 (SEQ ID NO: 4) is expressed under the control of the T3 promoter; the gIII gene on helper plasmid CCP2.1 (SEQ ID NO: 6) is expressed under the control of the T7 promoter; the gIII-neg gene on helper plasmid CCP3 (SEQ ID NO: 5) is expressed under the control of the T7 promoter; and the gIII-neg gene on helper plasmid CCP4 is expressed under the control of the T7 promoter. Among these, gIII-neg is a defective gIII, lacking approximately 70 amino acids between aa280 and aa350 of the gIII gene, and cannot support LWS proliferation and evolution. When gIII-neg is expressed together with gIII, it competitively inhibits LWS proliferation.

[0063] In this invention, the bacteriophage LWS and the helper plasmid CCP4 are the same as the bacteriophages SM and HP4 disclosed in application number 201610349254.3.

[0064] The mutagenic gene on mutagenic plasmid IMP1 is expressed under the control of the T7 promoter; unlike mutagenic plasmid IMP1, the mutagenic gene on mutagenic plasmid IMP2 is expressed under the control of the T3 promoter, and the gene sequence of mutagenic plasmid IMP2 is shown in SEQ ID NO: 7; unlike mutagenic plasmid IMP2, the mutagenic gene on mutagenic plasmid IMP3 is expressed under the control of the psp promoter. Phage M13-WT is a wild-type phage obtained by removing the insert gene from M13KO7 phage purchased from NEB. Compared to phage LWS, M13-WT (NCBI ACCESSION: V00604) and T7 phage (NCBI ACCESSION: NC_001604) have complete genomes and functions and can independently infect and proliferate in host bacteria.

[0065] Some of the bacteriophages and plasmids used in this invention were obtained by the inventors after the David R Liu laboratory provided some materials. Their genetic information has been reported in relevant literature (Nat Chem Biol. 2014 March; 10(3):216–222). Other plasmids and strains were constructed by the applicant. The host bacterium used in this invention is E. coli M15, which was obtained by introducing the F plasmid into E. coli MG1655 of the E. coli K12 series, with the genotype F'proA. + B + lacI q Δ(lacZ)M15 zzf::Tn10(Tet R ) / attB::aph tetR.

[0066] It should be noted that the host bacteria in this invention are not limited to E. coli M15; it can be any Escherichia coli carrying the F factor. Furthermore, E. coli M15, LWS, CCP1, CCP2, CCP2.1, CCP3, CCP4, IMP1, IMP2, and IMP3 in this invention can all be obtained by conventional molecular cloning methods such as PCR, enzyme digestion and ligation, and gene recombination, referring to gene maps and sequences. Gene recombination, PCR, and enzyme digestion and ligation are well-known techniques in the art, and it has been confirmed that corresponding strains, plasmids, and phages can be obtained. Therefore, the host bacteria, plasmids, and phages of this invention are reproducible and can be obtained by those skilled in the art using conventional methods. Accordingly, those skilled in the art should understand that this invention does not require strain preservation to satisfy the conditions of full disclosure.

[0067] In this invention, host bacteria carrying CCP1 and IMP1 are designated S1, host bacteria carrying IMP2, CCP2, and CCP3 are designated S2, and host bacteria carrying IMP2, CCP2, and CCP4 are designated S3. During positive selection, the initial LWS undergoes continuous directed evolution in S1 bacteria. The initial LWS carries the T7 RNAP gene and can only proliferate and evolve at a low level in S1 bacteria using the basal expression of CCP1. As the T7 RNAP carried by LWS continuously evolves towards T3 RNAP, an LWS mutant strain is generated that can initiate gIII expression controlled by the T3 promoter on CCP1. This mutant strain can further proliferate and evolve at a higher level in S1 bacteria, continuously improving its initiation activity for gIII controlled by the T3 promoter on CCP1, resulting in the mutant phage LWSe and the target gene evolved into pre-T3 RNAP (…). Figure 2 (a) This process is called positive screening.

[0068] After evolution, the pre-T3 RNAP on LWSe exhibits high activity against both T7 and T3 promoters. To improve the specificity of pre-T3 RNAP for the T3 promoter, it is necessary to perform evolutionary screening to identify T3 RNAPs with lower recognition and activation ability against the T7 promoter. This process is called negative selection.

[0069] Negative screening requires the use of CCP3 or CCP4. When LWSe, evolved through positive selection, infects S2 bacteria, the evolved target gene pre-T3RNAP on LWSe can initiate the expression of gIII-R5, inhibiting its proliferation. This situation continues until LWSe evolves some new phage mutants. These new phage mutants carry a target gene that can efficiently initiate the expression of the gIII gene controlled by the T3 promoter on CCP2, while having little or no initiation of the gIII-R5 gene expression controlled by the T7 promoter on CCP3 or CCP4. At this point, these new phage mutants continue to evolve further in S2 or S3 strains, eventually evolving into phage LWSeN carrying the highly specific T3 RNA polymerase gene T3RNAP. Figure 2 (b) CCP4 is a high-copy plasmid, expressing defective gene elements at a higher level. Therefore, CCP4 is an enhanced version of CCP3. The S3 host bacteriology is used to replace the S2 host bacteriology, and bacteriophages that have evolved in the S2 host bacteriology can be transferred to the S3 host bacteriology for further evolution.

[0070] The feasibility of the present invention is illustrated by the following embodiments. It should be noted that the following embodiments are merely exemplary and are intended only to illustrate the feasibility of the present invention, and are not intended to limit the scope of protection of the present invention.

[0071] Example 1 The following preparations should be made before conducting evolutionary experiments.

[0072] 1) Host bacteria S1 carrying CCP1 and IMP1 plasmids were cultured in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol at 37°C and 220 rpm until OD. 600 =0.3. Continue diluting S1 by 100 times and then incubate again under the same conditions until OD reaches 0.3. 600 =0.3. After two successful cultures, the host bacterium S1 can be used for evolutionary experiments.

[0073] 2) Host bacteria S2 carrying CCP2, CCP3, and IMP2 plasmids were cultured in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, 50 μg / ml carbenicillin, and 25 μg / ml chloramphenicol at 37°C and 220 rpm until OD. 600=0.3. Continue diluting S2 by 100 times and then incubate again under the same conditions until OD reaches 0.3. 600 =0.3. After two successful cultures, the host bacterium S2 can be used for evolutionary experiments.

[0074] 3) Host bacteria S3 carrying CCP2, CCP4, and IMP2 plasmids were cultured in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, 50 μg / ml carbenicillin, and 25 μg / ml chloramphenicol at 37°C and 220 rpm until OD. 600 =0.3. Continue diluting S3 by 100 times and then incubate again under the same conditions until OD reaches 0.3. 600 =0.3. After two successful cultures, the host bacterium S3 can be used for evolutionary experiments.

[0075] 4) The host bacterium S4 carrying CCP2 was cultured in LB medium containing 50 μg / ml tetracycline and 50 μg / ml carbenicillin at 37°C and 220 rpm until OD. 600 =0.3. Continue diluting S4 by 100 times and then incubate again under the same conditions until OD reaches 0.3. 600 =0.3. After two successful incubations, the host bacterium S4 can be used for experiments.

[0076] 5) The host bacterium S5 carrying CCP2.1 was cultured in LB medium containing 50 μg / ml tetracycline and 50 μg / ml carbenicillin at 37°C and 220 rpm until OD. 600 =0.3. Continue diluting S5 by 100 times and then incubate again under the same conditions until OD reaches 0.3. 600 =0.3. After two successful incubations, the host bacterium S5 can be used for experiments.

[0077] 6) The host bacterium S6 carrying CCP1 was cultured in LB medium containing 50 μg / ml tetracycline and 50 μg / ml spectinomycin at 37°C and 220 rpm until OD. 600 =0.3. Continue diluting S6 by 100 times and then incubate again under the same conditions until OD reaches 0.3. 600 =0.3. After two successful cultures, the host bacterium S6 can be used for evolutionary experiments.

[0078] 7) The host bacteria S7 carrying IMP3 and CCP1 were cultured in LB medium containing 50 μg / ml tetracycline, 50 μg / ml spectinomycin, and 25 μg / ml chloramphenicol at 37°C and 220 rpm until OD. 600 =0.3. Continue diluting S7 by 100 times and then culturing again under the same conditions until OD reaches 0.3. 600=0.3. After two successful incubations, the host bacterium S7 can be used for experiments.

[0079] 8) E. coli M15 was cultured in LB medium containing 50 μg / ml tetracycline at 37°C and 220 rpm until OD200. 600 =0.3. Continue with E. coli After diluting M15 by 100 times, it was cultured again under the same conditions until OD. 600 =0.3. After two cultivation cycles, E. coli M15 can be used for experiments.

[0080] 9) E. coli MG1655 was cultured in LB medium at 37°C and 220 rpm until OD. 600 =0.3. Continue with E. coli After diluting MG1655 by 100 times, it was cultured again under the same conditions until OD. 600 =0.3. After two cultivation cycles, E. coli MG1655 can be used for experiments.

[0081] Example 2 Method for observing LWS phage plaques in S4 or S5 host bacteria 1) Spread 10 ml of 1.5% agarose gel into a 10 cm bacterial culture plate and let it solidify at room temperature for 20 minutes.

[0082] 2) When the concentration of phage LWS is unknown, the LWS needs to be adjusted according to 10... 1，2，3，4，5，6，7 Perform continuous gradient dilution at multiples of 1:1.

[0083] 3) Take several groups of 200 μL of the S4 or S5 host bacteria prepared in "Example 1", add 10 μL of LWS at different dilution gradients in "2)" to each group, then add 4 ml of LB medium containing 0.4% bacteriological agar stored at 55℃ and carbenicillin at a final concentration of 50 μg / ml. After mixing with a vortex mixer, spread the sample onto the plate prepared in "1)". Let it stand at room temperature for 1 hour to allow it to solidify.

[0084] 4) Incubate overnight in a 37°C biochemical incubator.

[0085] 5) Observe and count the number of plaques formed in each gradient dilution of the sample, and calculate the LWS concentration of the original sample.

[0086] The CCP2.1 gIII gene carried by S5 is promoted by the T7 promoter, and the LWS phage carrying the T7 RNAP target gene can dilute plaques in the S5 host bacteria. In contrast, the CCP2 gIII gene carried by S4 is promoted by the T3 promoter, and only LWSe carrying the target gene that has evolved T3 RNAP activity can form plaques in the S4 host bacteria. By comparing the changes in the number of plaques formed by LWSe in the S4 and S5 host bacteria, the evolutionary effect of LWSe can be directly analyzed.

[0087] Example 3 Host bacteria, bacteriophage infection and motion test 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. Let it stand at room temperature for 1 hour to allow it to solidify.

[0088] 2) such as Figure 1 As shown in Figure a, 2 μl of the host bacterium S5 prepared in "Example 1" was inoculated onto the central surface of the plate. 2 μl of a storage concentration of 10 was then inoculated at each of the three corners 1 cm outside the S5 inoculation site. 6 LWS phage pfu / ml.

[0089] 3) The plates were incubated overnight in a 37°C biochemical incubator. Afterwards, the following observations could be made: Figure 1 The phenomenon shown in b. The host bacteria move from the center to the edge. During this movement, the host bacteria come into contact with the bacteriophage, become infected, and produce progeny bacteriophages, while continuing to move outwards. Because the infected host bacteria grow slowly, their numbers are relatively small; while in the uninfected area, the host bacteria maintain their original state, and their numbers are relatively large. Therefore, a transparent V-shaped infection patch with fewer bacteria can be seen in the infected area. The presence of this transparent infection patch indicates the presence of bacteriophage infection, and the transparency and size of the transparent patch represent the infective activity of the bacteriophage.

[0090] Example 4 SPACE is undergoing positive selection and evolution. 1) First round of evolution: Prepare three 10cm bacterial culture plates (a, b, and c). Add 10ml of LB medium containing 0.25% bacteriological agar to each plate. The medium contains 50μg / ml tetracycline and 50μg / ml spectinomycin. Incubate at room temperature for 1 hour to allow solidification.

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

[0092] 2) such as Figure 1 As shown in Figure a, 2 μl of the host bacterium S6 prepared in "Example 1" was inoculated onto the central surface of three plates a, b, and c. At the three corners of plate a, 1 cm from the inoculation point of S6, 2 μl of a storage concentration of 10... 8 LWS phage at pfu / ml was inoculated at three corners of plate b, 1 cm from the inoculation site at S6, with a storage concentration of 10 pfu / ml. 9 LWS phage at pfu / ml was inoculated at three corners of plate c, 1 cm from the inoculation site at S6, with a storage concentration of 10 pfu / ml. 10 LWS phage pfu / ml.

[0093] 2 μl of the host bacterium S1 prepared in “Example 1” was inoculated onto the central surface of four plates d, e, f, and g. 2 μl of a storage concentration of 10⁻⁶ was inoculated at each of the three corners of plate d, 1 cm from the inoculation point of S1. 8 LWS phage at pfu / ml was inoculated at three corners of an e-plate, 1 cm from the S1 inoculation site, with a storage concentration of 10 pfu / ml. 9 LWS phage at pfu / ml was inoculated at three corners of the f plate, 1 cm from the S1 inoculation site, with a storage concentration of 10 pfu / ml. 10 LWS phage at pfu / ml. No phage inoculation on g plates.

[0094] Plates d, e, and f represent SAPCE evolution groups with different amounts of initial phage added; plates a, b, and c represent control groups without the mutagenic plasmid IMP1 at different phage addition amounts; plate g represents the negative control group without phage.

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

[0096] 4) The effects of a~g plate culture are as follows Figure 4 As shown in a~g. In plates a, b, and c, the S6 host bacteria did not carry mutagenic plasmids, and the bacteriophage could not undergo mutational evolution. On plates a, b, and c, bacteriophage LWS could only rely on the background expression of CCP1 to undergo weak proliferation, and was quickly shed by the moving host bacteria, insufficient to form visible infection plaques. The final result was the same as the negative control in group g.

[0097] In plates d, e, and f, the host bacterium S1 carries the mutagenic plasmid IMP1. Therefore, although phage LWS initially can only replicate and proliferate using the background expression of CCP1, phage LWS can activate the induced mutation gene on IMP1, helping itself to mutate and evolve T3RNAP activity. Thus, infection plaques can quickly form on plates d, e, and f, and the larger the initial amount of phage added, the more prominent the infection plaques.

[0098] The infection patches were obvious on plate e, and no nonspecific infection patches were observed on plate b, which contained the same amount of initial phage LWS as plate e. Therefore, plate e was selected for experimental analysis in subsequent implementation cases.

[0099] The evolved bacteriophage LWSe was found in the infection plaques that evolved on e-plates. 5 μl samples were taken from the starting point α (near the center of the plate), the midpoint β, and the ending point γ (far from the center of the plate) of the infection plaques on e-plates, and labeled LWSeα, LWSeβ, and LWSeγ, respectively. The samples were diluted 100-fold with LB liquid medium and vortexed for 2 min. After filtration through a 0.22 μm filter, the samples were analyzed according to the method described in "Implementation Case 2" to detect the formation of T3 plaques (LWSeα, LWSeβ, LWSeγ) in S4 host bacteria (where gIII gene expression is controlled by the T3 promoter) and the formation of T7 plaques (LWSeγ) in S5 host bacteria (where gIII gene expression is controlled by the T7 promoter).

[0100] like Figure 4 As shown in figure h, the pre-evolutionary bacteriophage LWS carried the wild-type T7 RNAP gene and could only form T7 plaques in host bacterium S5, but could not form T3 plaques in host bacterium S4. With evolution, bacteriophage LWS evolved into bacteriophage LWSe. The target gene carried on LWSe continuously mutated, enabling it to acquire T3 RNAP activity, recognize the T3 promoter, and form T3 plaques in host bacterium S4. Moreover, the farther away from the center of the plate and the longer the evolutionary time, the better the evolutionary effect, and the more pronounced the T3 RNAP activity acquired by LWSe. Figure 4 As shown in h, the ability of LWS to form T3 plaques in S4 increases progressively from LWS to LWSeγ. Therefore, in subsequent implementation cases, samples from the endpoint of the infection plaque furthest from the center of the plate after each evolution were diluted 100-fold, filtered through a 0.22 μm filter, and then analyzed or further evolved.

[0101] To maintain consistency with subsequent implementation examples, the LWSeγ sample will be relabeled as LWSe1. The "1" represents one evolutionary round. Therefore, in this invention, LWSe1 and LWSeγ represent the same sample's name in different scenarios.

[0102] Second round of evolution: On the same plate, S1 host bacteria were inoculated in the center of the plate, and 2 μl of filtered bacteriophage LWSe1 was inoculated in each of the three corners 1 cm away from the S1 inoculation site. The second round of evolution was carried out under the same conditions.

[0103] Five μl of LWSe2 phage sample from the endpoint of the second round of evolutionary infection plaques was diluted 100-fold and filtered through a 0.22 μm filter. The formation of T3 plaques in the S4 host bacterium and T7 plaques in the S5 host bacterium were then detected. Figure 5 As shown, after an additional round of evolution, LWSe2 has a stronger ability to form plaques T3plaques in the S4 host bacteria than LWSe1.

[0104] Example 5 SPACE negative selection evolution under S2 or S3 host bacteria 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. Incubate at room temperature for 1 hour to allow solidification. In addition to the T7 promoter, the CCP3 and CCP4 plasmids carried by S2 also have a theophylline-induced riboswitch. However, this riboswitch is not precise; even without theophylline, there is a certain background expression of gIII-neg, which inhibits the proliferation of phage LWSe carrying the target gene and possessing wild-type T7 RNAP activity.

[0105] Two μl of host bacteria S2 was inoculated into the center of the plate, and then two μl of filtered bacteriophage LWSe2 was inoculated into each of the three corners 1 cm away from the S2 inoculation site. The plate was then incubated overnight at 37°C. This was the third round of evolution.

[0106] Take 5 μl of LWSe3 phage sample from the endpoint of the third round of evolutionary infection plaques, dilute it 100 times, filter it through a 0.22 μm filter, and then detect the formation of phage plaque T3 plaques in S4 host bacteria and phage plaque T7 plaques in S5 host bacteria, respectively.

[0107] 2) Fourth round of evolution: The filtered LWSe3 sample was subjected to the fourth round of evolution under the same conditions as in step “1)” of this implementation case. Similarly, 5 μl of LWSe4 phage sample from the endpoint of the fourth round of evolution was diluted 100 times and filtered through a 0.22 μm filter. The formation of T3 plaques in the S4 host bacteria and the formation of T7 plaques in the S5 host bacteria were then detected.

[0108] 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. Incubate at room temperature for 1 hour to allow solidification. The addition of theophylline can increase the expression of CCP3 and CCP4gIII-neg, enhancing the evolutionary selection pressure.

[0109] Two μl of host bacteria S2 was inoculated into the center of the plate, and then two μl of filtered bacteriophage LWSe4 was inoculated into each of the three corners 1 cm away from the S2 inoculation site. The plate was then incubated overnight at 37°C. This was the fifth round of evolution.

[0110] Take 5 μl of LWSe5 phage sample from the endpoint of the fifth round of evolutionary infection plaques, dilute it 100 times, filter it through a 0.22 μm filter, and then detect the formation of phage plaque T3 plaques in S4 host bacteria and phage plaque T7 plaques in S5 host bacteria, respectively.

[0111] 4) Sixth 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. Incubate at room temperature for 1 hour to allow it to solidify.

[0112] Two μl of host bacteria S3 was inoculated into the center of the plate, and then two μl of filtered bacteriophage LWSe5 was inoculated into each of the three corners 1 cm away from the S3 inoculation site. The plate was then incubated overnight at 37°C. This was the sixth round of evolution.

[0113] Take 5 μl of LWSe6 phage sample from the endpoint of the sixth round of evolutionary infection plaques, dilute it 100 times, filter it through a 0.22 μm filter, and then detect the formation of phage plaque T3 plaques in S4 host bacteria and phage plaque T7 plaques in S5 host bacteria, respectively.

[0114] 5) Seventh 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. Incubate at room temperature for 1 hour to allow it to solidify.

[0115] Two μl of host bacteria S3 was inoculated into the center of the plate, and then two μl of filtered bacteriophage LWSe6 was inoculated into each of the three corners 1 cm away from the S3 inoculation site. The plate was then incubated overnight at 37°C. This was the sixth round of evolution.

[0116] Take 5 μl of LWSe7 phage sample from the endpoint of the seventh round of evolutionary infection plaques, dilute it 100 times, filter it through a 0.22 μm filter, and then detect the formation of phage plaque T3 plaques in S4 host bacteria and the formation of phage plaque T7 plaques in S5 host bacteria, respectively.

[0117] The effects of 7 rounds of evolution are as follows Figure 5 As shown. Among them. Figure 5 'a' represents the copy number of different CCPs. The copy number affects the gene expression level and the strength of selection pressure during evolution. Figure 5 b represents the effect of each round on the board, where the row of evolutionary pressure is filled with colors from light to dark to indicate the evolutionary selection pressure from weak to strong brought about by different evolutionary conditions. Figure 5 c. The effects of each round of evolution on plaque formation in S4 and S5 host bacteria. As shown in the figure, with the increase of the number of evolutionary rounds, the selection pressure of the evolutionary process becomes stronger and the evolutionary effect becomes more obvious. LWS phage carrying the wild-type T7 RNAP gene can only form T7 plaques in S5 host bacteria where the expression of the gIII gene is controlled by the T7 promoter, but cannot form T3 plaques in S4 host bacteria where the expression of the gIII gene is controlled by the T3 promoter. As evolution progresses, from LWSe1 to LWSe7 phages, their ability to form T7 plaques in S5 host bacteria generally decreases, but their ability to form T3 plaques in S4 host bacteria generally increases.

[0118] Example 6 SPACE product sequencing analysis Several clones of each of the first two rounds of evolutionary products, phages LWSe1 and LWSe2, were purified and sequenced for the target gene. For example... Figure 5As shown in d, the target genes they carry all have corresponding mutations directly in amino acids 1-310 at the N-terminus. Existing reports indicate that this region is related to T7 RNAP recognition of the promoter. Among them, the E222K mutation has been reported by David R. Liu to affect the specificity of T7 RNAP.

[0119] Comparing the plaque-forming ability and sequencing results of bacteriophages before and after evolution in S5 and S4 host bacteria, it can be seen that the change in the activity of the target gene carried by LWSe bacteriophage from T7RNAP to T3RNAP is caused by mutations resulting from the evolution of SAPCE.

[0120] Experimental Example 1 IMP3-induced SPACE evolution test 1) Prepare three 10cm bacterial culture plates (a, b, and c). Add 10ml of LB medium containing 0.25% bacteriological agar to each plate. The medium contains 50μg / ml tetracycline, 50μg / ml spectinomycin, and 25μg / ml chloramphenicol. Incubate at room temperature for 1 hour to allow it to solidify.

[0121] 2) such as Figure 1 As shown in Figure a, 2 μl of the host bacterium S7 prepared in Example 1 was inoculated onto the central surface of plates a and c, respectively. At the three corners 1 cm away from the inoculation site of the host bacterium S7 on plate a, 2 μl of a storage concentration of 10 was inoculated onto each of the plates. 9 LWS phage at pfu / ml. Plate c, as the negative group, was not inoculated with phage.

[0122] 2 μl of the host bacterium S1 prepared in Case 1 was inoculated onto the central surface of plate b. 2 μl of a storage concentration of 10 was then inoculated at each of the three corners 1 cm outside the inoculation site of the host bacterium S1 on plate b. 9 LWS phage pfu / ml.

[0123] 3) Place the plates in a 37°C biochemical incubator and incubate overnight.

[0124] 4) The effects of the evolution of the a~c tablets are as follows: Figure 6 As shown in Figure ac, host bacteria S7 carrying IPM3 and host bacteria S1 carrying IMP1 exhibited similar evolutionary plaques. 5 μl of evolved phage sample LWSeP1 was taken from the endpoint of the evolutionary plaques in host bacteria S7, diluted 100-fold, and filtered through a 0.22 μm filter. The formation of plaques T3 in host bacteria S4 and plaques T7 in host bacteria S5 were then analyzed.

[0125] Five μl of evolved phage sample LWSeT1 was taken from the endpoint of the evolutionary infection plaque in host bacteria S1 group, diluted 100-fold, and filtered through a 0.22 μm filter. The formation of phage plaque T3 in host bacteria S4 and phage plaque T7 in host bacteria S5 were then detected.

[0126] like Figure 6 As shown in d, the number of T3 and T7 plaques of LWSeT1 and LWSeP1 are similar. This indicates that the IMP3 and IMP1 controlled by the psp operon have the same promoting effect on mutational evolution.

[0127] Therefore, IMP3 can be directly used to replace IMP1 and IMP2, allowing IMP expression to be decoupled from the target gene's activity. In this case, regardless of whether the target gene is synthesized through evolution, or whether it's a positive or negative screening process, the same IMP3 can be used.

[0128] Experiment Example 2 Vicious bacteriophage infection exercise test 1) The above embodiments were based on chronic infection with M13 bacteriophage. However, the present invention can also be used to conduct evolutionary experiments on other bacteriophages. Here, the virulent bacteriophage T7 is used as an example for illustration.

[0129] 2) Add 10 ml of LB medium containing 0.25% bacteriological agar to a 10 cm bacterial culture plate and let it solidify at room temperature for 1 hour.

[0130] 3) such as Figure 1 As shown in Figure a, 2 μl of the host bacteria prepared in "Implementation Case 1" is added. E. coli MG1655 was inoculated onto the central surface of the plate. At a distance... E. coli 2 μl of MG1655 was inoculated at each of the three corners 1 cm outside the inoculation site, with a storage concentration of 10. 4 10 5 10 6 T7 phage at pfu / ml. The control group used LB medium instead of phage.

[0131] 4) The plates were incubated overnight in a 37°C biochemical incubator. Afterwards, the following observations could be made: Figure 7 The phenomenon shown in b is as follows: The host bacteria move from the center to the edge. During this movement, the host bacteria come into contact with the phage, become infected, and produce progeny phages, while continuing to move outwards. T7 phage is a virulent phage; all infected host bacteria lyse and die. Therefore, a V-shaped clear plaque is formed on the plate. The 16a control group, lacking phages, does not form a V-shaped plaque; its bacteria are evenly distributed in a circular pattern. The inoculum size is 10... 6 The pfu / ml group, in Figure 7Samples were taken from point α in the infected area, point β in the uninfected area, and point γ in the junction area, and then, according to "Example 2", using... E. coli MG1655 was used for plaque counting. For example... Figure 7 As shown in c, 10 was measured at point γ. 9 T7 phage has a concentration on the order of pfu / ml. It is evident that virulent T7 phage can also move and infect on plates along with the host bacteria, forming visible infection plaques as markers of infection. Therefore, the embodiments of this invention performed on M13 phage can be performed entirely on T7 phage.

[0132] The above embodiments illustrate the present invention by using the evolution of the T7 RNA polymerase gene T7RNAP, which recognizes the T7 promoter, into the T3 RNA polymerase gene T3RNHP, which recognizes the T3 promoter. By replacing the T7RNHP gene in the LWS with other target genes (such as protease genes, cellulase genes, fluorescent protein genes, density sensing genes, etc.) and adjusting the expression regulation and post-expression modification of the gIII and gIII-R5 genes in the CCP1, CCP2, CCP3, and CCP4 plasmids accordingly, the expression of gIII and gIII-R5 is bound to the biological activity of the new target gene to be evolved on the LWS. This allows the system to be used for directed evolution of the new target gene.

[0133] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims.

Claims

1. A visual, continuous spatial directed evolution method, characterized in that, The host grows and moves in a solid culture space, the host carries an exogenous target gene to be evolved, the host itself contains gene elements that assist the evolution of the target gene, and the target gene is associated with the growth and movement of the host; Through the host's growth process, different spatial distribution patterns are formed in the solid culture space, and evolutionary products are obtained by screening.

2. The visual, continuous spatial directed evolution method according to claim 1, characterized in that, The target gene is located in the host genome or plasmid or in a parasitic organism corresponding to the host.

3. The visual, continuous spatial directed evolution method according to claim 1, characterized in that, The parasitic organisms include any one of bacteriophages, algaeophages, animal and plant viruses, fungal viruses, mycoplasma, chlamydia, and bacteria.

4. The visual, continuous spatial directed evolution method according to claim 1, characterized in that, The parasitic organism is a bacteriophage; The host is any one of the following: The natural host bacteria of the non-deficient strain of the bacteriophage; The strain obtained by genetically modifying the natural host bacteria of the non-deficient strain of the bacteriophage; Non-natural host bacteria that acquire susceptibility through genetic modification; Furthermore, the hosts include Escherichia coli, Pasteurella, Shigella, Pseudomonas, Xanthomonas, Salmonella, Staphylococcus aureus, and genetically modified strains that alter their susceptibility; Preferably, the host is *Escherichia coli* carrying factor F. Furthermore, the bacteriophage is a temperate bacteriophage, a virulent bacteriophage, or a chronically infectious bacteriophage; Furthermore, the bacteriophages include filamentous bacteriophages, T4 bacteriophages, T7 bacteriophages, λ bacteriophages, P1 bacteriophages, P2 bacteriophages, and P22 bacteriophages. X174 bacteriophage, SP6 bacteriophage; Preferably, the filamentous phage includes M13 filamentous phage and f1 filamentous phage.

5. The visual, continuous spatial directed evolution method according to claim 1, characterized in that, The target gene is a combination of one or more protein-coding genes and non-coding genes; Furthermore, the target gene is selected from one or more of the following: T7 RNA polymerase gene, protease gene, cellulase gene, fluorescent protein gene, and density sensing gene.

6. The visual, continuous spatial directed evolution method according to claim 1, characterized in that, The gene element that assists in the evolution of the target gene is a mutation-inducing plasmid, and the mutation-inducing plasmid is initiated or induced to express by the target gene before and after evolution, respectively.

7. The visual, continuous spatial directed evolution method according to claim 6, characterized in that, The induced mutation plasmid contains a mutagenic gene selected from at least one of the following: DNAQ926 gene (a DNAQ gene mutant with amino acids mutated to Ala at positions 12 and 14), deoxyadenosine methyltransferase dam gene, hemimethylated GATC-binding protein seqA gene, activation-induced cytosine deaminase gene AID, uracil DNA glycosyltransferase inhibitor gene Ugi from phage PBS2, and transcriptional repressor emrR.

8. The visual, continuous spatial directed evolution method according to claim 1, characterized in that, The solid culture space includes a two-dimensional planar culture structure and a three-dimensional spatial culture structure; Furthermore, the vertical movement and evolution of the solid culture space is maintained by periodically assembling a solid culture system; Furthermore, the directed evolution is high-throughput evolution; Furthermore, the high-throughput evolution is achieved by using multiple sets of solid culture spaces or at different locations within the solid culture space.

9. A visual, continuous spatial directed evolution method according to any one of claims 4-8, characterized in that, The target gene is associated with the host growth and movement via a helper plasmid, which contains at least a first helper plasmid, which is either helper plasmid CCP1 or helper plasmid CCP2. The nucleic acid sequence of helper plasmid CCP1 is shown in SEQ ID NO: 3, and the nucleic acid sequence of helper plasmid CCP2 is shown in SEQ ID NO:

4. Furthermore, the helper plasmid also includes a second helper plasmid, which is helper plasmid CCP3 or helper plasmid CCP4; The nucleic acid sequence of the helper plasmid CCP3 is shown in SEQ ID NO:

5.

10. A visual, continuous spatial directed evolution method according to claim 9, characterized in that, The directed evolution is carried out by succession of different hosts, and the gene elements supporting phage proliferation in the later host include helper plasmids that support the proliferation of the evolved phage and helper plasmids that inhibit the proliferation of the pre-evolutionary phage.

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