CN114934059B - Method for simplifying phage genome framework in high flux - Google Patents

Abstract

The application relates to the technical field of life science and provides a method for simplifying a phage genome framework in high flux. The method comprises the following steps: designing and synthesizing a CiPGr sequence library of the phage, and assembling the CiPGr sequence library with a pTarget plasmid skeleton to form a CiPGr plasmid library; transforming the CiPGr plasmid and extracting the CiPGr plasmid; transforming the CiPGr plasmid into host bacteria containing the spCas9 plasmid to obtain a CiPGr plasmid-bacteria library; culturing wild phage and corresponding CiPGr plasmid-bacterial library in culture medium, and performing iterative culture to generate mutant phage library; sequencing the mutant phage library obtained after a certain transfer times to confirm all the deletable genes in the phage. The method provided by the application can realize the genome simplification of all phages including tailed phages.

Description

A method for high-throughput simplification of the phage genome backbone

technical field

This application relates to the technical field of life sciences, in particular to a method for simplifying the skeleton of a phage genome with high throughput.

Background technique

Phages are the most abundant and genetically diverse organisms on Earth. For more than a century, phage research has been key to many biological discoveries, providing important biotechnological tools for molecular biology. In recent years, viral metagenomic sequencing has revealed a large number of phage sequences found in the human gut and other environments. The majority (>75%) of these sequences were novel, and more than 95% of the sequences belonged to tailed double-stranded (ds) DNA phages. In addition, phages have been considered as potential natural antibacterial agents for the treatment of bacterial infections. But at the same time, as a potential natural antibacterial drug for the treatment of bacterial infections, phages have problems such as host specificity and easy resistance, which directly affect the druggability of phages. Consequently, many synthetic biology efforts have been conducted on phages to overcome these limitations.

Tailless phages, such as M13 and X174, have very compact and small genomes (<10 kb), which are relatively simple to encode a limited number of genes, making them easy to edit and understand the function of their genes. However, the genomes of tailed phages are usually relatively large (14-500 kbp) and extremely diverse, which makes it challenging to obtain simplified genomes of tailed phages on a large scale. One of the challenges is that there is no efficient way to identify non-essential genes of phages at the genome scale. For example, the 25 non-essential genes of the model bacteriophage T7 were obtained from the knowledge accumulated in a large number of studies in the last three to four decades. The amplification of bacteriophage is completely dependent on its host, and its unique self-propagating properties make the methods widely used in bacteria (simplified genome methods widely used in microorganisms such as Escherichia coli, yeast, Bacillus and mycoplasma, such as homologous recombination, Tn5 mutations, etc.) may not be suitable for large-scale application in tailed phages. In addition, due to the highly diverse characteristics of phage genomes, it is difficult to obtain non-essential gene information through comparison of bioinformatics methods.

De novo synthesis of a simplified genome requires high-throughput identification of non-essential genes of phages, but high-throughput identification methods such as the dCas9 method fail for two reasons: First, because the phage genome will replicate many copies in bacteria, many phage genomes Second, many phage genes are transcribed in tandem, and dCas9 inhibits upstream genes, which will lead to the inhibition of expression of all downstream genes, resulting in false negative results. The transposon method similar to Tn5 also leads to a decrease in identification efficiency due to the unique growth mode of phage.

Contents of the invention

The purpose of this application is to provide a high-throughput method for simplifying the skeleton of the phage genome, aiming at solving the problems of complexity and low efficiency of the method for simplifying the phage genome.

In order to realize the above-mentioned application purpose, the technical scheme adopted in this application is as follows:

A high-throughput method for simplifying the phage genome backbone, comprising the steps of:

Designing and synthesizing multiple CiPGr sequence libraries of two or more phages, assembling each of the CiPGr sequence libraries with the pTarget plasmid backbone to form a CiPGr plasmid library; wherein the CiPGr sequence library includes n CiPGr sequences, n is a natural number greater than or equal to 2, the CiPGr sequence comprises gRNA, and the gRNA of each CiPGr sequence is different;

Transforming the CiPGr plasmid in the CiPGr plasmid library, screening transformed cells containing the CiPGr plasmid, extracting the CiPGr plasmid from the transformed cell; transforming the CiPGr plasmid into a host bacterium containing the spCas9 plasmid In, the CiPGr plasmid-bacteria library is obtained, and the CiPGr plasmid-bacteria library contains n kinds of CiPGr plasmid-bacteria;

Cultivate the wild-type phage and the corresponding CiPGr plasmid-bacteria library in the culture medium, transfer the mutant phage product to a fresh CiPGr plasmid-bacteria library to continue culturing, and repeat iterative cultivation to generate a mutant phage library;

Sequence the mutant phage library obtained after a certain number of transfers, and confirm all the deleteable genes in the phage.

In one embodiment, the steps of generating the mutant phage library are:

Cultivate the wild-type phage and the corresponding CiPGr plasmid-bacterial library in a culture medium to generate a first-generation mutant phage product, centrifuge the first-generation mutant phage product, and take the supernatant to obtain a first-generation mutant phage product. Phage library; part of the first-generation mutant phage library and the fresh CiPGr plasmid-bacteria library medium are continued to be cultivated to generate second-generation mutant phage products, and the second-generation mutant phage products are centrifuged to take the supernatant solution to obtain the second-generation mutant phage library; part of the second-generation mutant phage library and the fresh CiPGr plasmid-bacteria library are cultivated in a medium to generate a third-generation mutant phage product, and the third-generation mutant phage product is centrifuged. Mutate the phage product, and take the supernatant to obtain the third-generation mutant phage library; repeat the step of iterative culture, so that the number of transfers reaches 300-600 times, and collect each mutant phage library.

In one embodiment, the method further comprises screening for a single active mutant phage.

In one embodiment, the screening for a single active mutant phage comprises:

Cultivate 1×10 ³ -5×10 ³ PFU mutant phage library and wild-type host cells in solid medium, randomly select several large plaques and small plaques for isolation and purification; host cells cultured overnight and fresh LB The culture medium is added to a 96-well plate, and after adding a single mutant phage, it is cultured on a microplate reader, and the OD ₆₀₀ is detected every once in a while for 12 to 24 hours; the mutant phage is selected according to the bactericidal curve, and the bacteriophage is selected by the drawing method. The selected mutant phages were further purified.

In one embodiment, the method also includes screening dominant mutant phages, the steps comprising:

After mixing the mutant phages in the mutant phage library, co-culture with wild-type host cells, transfer to fresh wild-type host cells for re-cultivation, and repeat the step of "transferring to fresh wild-type host cells for re-culture" N-1 times to get TN mutant phage;

Mix 1×10 ³ -5×10 ³ PFU of TN mutant phage, wild-type host cells and LB agar, pour into LB plates, and culture overnight;

After selecting different plaques for purification, overnight cultured host cells and fresh LB medium were added to a 96-well plate, and a single plaque was purified and transferred to a 96-well plate, cultured on a microplate reader, and Test the OD ₆₀₀ at intervals for 12-24 hours; screen the dominant mutant phages according to the bactericidal curve.

In one embodiment, the method further comprises the determination of non-essential genes, quasi-essential genes and essential genes in the phage.

In one embodiment, the determination of non-essential genes, quasi-essential genes and essential genes in the phage includes:

Analyzing the probability of the deletion gene of the mutant phage in the mutant phage library, the deletable gene with a deletion frequency of <5% is determined as a quasi-essential gene of the phage, and the deletable gene with a deletion frequency of >5% is determined as a non-essential gene of the phage. Essential genes, genes for which no deletions were detected were identified as essential genes for the phage.

In one embodiment, the CiPGr sequence further comprises a barcode, two homology arms, a promoter and primers.

In one embodiment, after the CiPGr plasmid in the CiPGr plasmid library is transformed, the transformed cells containing the CiPGr plasmid are screened, and the CiPGr plasmid is extracted from the transformed cells, including:

Transform the CiPGr plasmid in the CiPGr plasmid library into DH5α Escherichia coli competent cells, transform in parallel 2 to 10 times, and collect the transformed DH5α Escherichia coli competent cells; spread the DH5α Escherichia coli competent cells on cultured on a petri dish containing the CiPGr plasmid, and the DH5α Escherichia coli transformed cells containing the CiPGr plasmid were screened, and the CiPGr plasmid was extracted from the DH5α Escherichia coli transformed cell.

In one embodiment, prior to the step of assembling all the CiPGr sequences in the CiPGr sequence library with the pTarget plasmid backbone, PCR separation of the CiPGr sequences in the CiPGr sequence library according to different phages is also included.

In one embodiment, the cycle condition of the PCR separation is:

98°C, 2 minutes; 98°C, 10s, 58°C, 20s, 72°C, 6s; 72°C, 10 minutes, store at 4°C.

In one embodiment, the method of assembling the CiPGr sequence library with the pTarget plasmid backbone is as follows: after connecting the CiPGr sequence library and the pTarget plasmid backbone through Gibson assembly reaction, then purifying.

In one embodiment, before the step of culturing the wild-type phage and the corresponding CiPGr plasmid-bacteria library in the culture medium, it also includes: adding the CiPGr plasmid-bacteria library to the LB medium, adding antibiotics and L-arabinose overnight culture.

In one embodiment, the number of repetitions of the iterative culture is 300-600 times.

In one embodiment, in the step of repeated iterative cultivation, the titrated concentration of the mutant phage library is regularly determined; when the concentration of the obtained mutant phage is less than 10 ⁵ /ml, wild-type host cells are used to cultivate the mutant phage library.

In one embodiment, the phage is tailed phage, tailless phage or other eukaryotic viruses.

As an embodiment, the phage is replaced by a eukaryotic virus, the host bacterium is replaced by a host cell, and the CiPGr plasmid-bacteria is replaced by a CiPGr plasmid-cell.

The beneficial effect of the high-throughput method for simplifying the phage genome skeleton provided by the embodiment of the present application is: for the deletable gene of the phage, a CiPGr sequence library with different gRNAs in the CiPGr sequence is designed, and the CiPGr sequence is assembled with the pTarget plasmid backbone to form CiPGr plasmid (i.e., pTarget plasmid), the CiPGr plasmid is transformed into the host bacteria containing the spCas9 plasmid to obtain the CiPGr plasmid-bacterial library. At this time, the host bacteria contain both the pTarget plasmid and the spCas9 plasmid, and different individual bacteria in the CiPGr plasmid-bacteria library may contain different pTarget plasmids. Infect the CiPGr plasmid-bacteria with wild-type phage, guide the guide RNA (gRNA) encoded by pTarget to guide the Cas9 nuclease encoded by spCas9 to bind to the target gene, and make the target gene double-strand break, and induce homologous recombination through homologous sequences (HR) repair, resulting in gene deletion or disruption. If the gene is not essential for phage growth, the offspring of the mutant phage can amplify without the gene. Through iterative culture, phages infect different CiPGr plasmid-bacteria, resulting in the deletion of different genes, thereby continuously producing mutant phages. Mutant phages are continuously transferred to new double-plasmid-containing bacteria, namely CiPGr plasmid-bacteria, which may infect host bacteria containing different pTarget plasmids and delete different genes. The continuous high-throughput deletion of genes from top to bottom will continuously accumulate gene deletions on the phage genome, and obtain a mutant phage library containing different deletion genes. Sequence the mutant phages obtained after a certain number of transfers at intervals until the phage genome is obtained skeleton. This method enables rapid high-throughput simplification of the genome backbone of all phages, including tailed and tailed phages.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings that need to be used in the descriptions of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only of the present invention. For some embodiments, those of ordinary skill in the art can also obtain other drawings based on these drawings without paying creative efforts.

Figure 1 is a schematic flow diagram of the method for the high-throughput simplified phage genome backbone provided by the embodiment of the present application;

Fig. 2 is a schematic flow diagram of the method for the high-throughput simplified phage genome backbone provided by the embodiment of the present application;

Figure 3 is the phage deletable gene library provided in Example 1 of the present application;

Fig. 4 is the gel electrophoresis graph of gp4.7 and gp5.3 in the phage T7 genome in different transfer times among the mutants provided in Example 1 of the present application;

Fig. 5 is the phage plaque of the mutant phage T7 in the double-layer agar plate provided by Example 1 of the present application;

Fig. 6 is the bactericidal curve of mutant phages T7 and T4 provided in Example 1 of the present application to MG1655;

Fig. 7 is the bactericidal curve of the phage with stronger antibacterial ability than the wild type provided by Example 1 of the present application;

Fig. 8 is a gene map of the stronger mutant phage provided in Example 1 of the present application.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects to be solved in the present application clearer, the present application will be further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application, and are not intended to limit the present application.

The terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of technical features. "Plurality" means two or more, unless otherwise clearly and specifically defined.

Phage synthetic biology can integrate functional genes or gene circuits into the phage genome to enhance its antibacterial activity and potential in phage therapy and other different bioengineering applications. Such gene circuit integration can be challenging due to the limited space for DNA packaging of phage particles. A phage's accessory gene can help a phage better adapt to a wide range of ecological settings, but it may not be necessary for a given situation. Deletion of these non-essential genes can create some space in the phage genome. At the same time, the removal of redundant genes from the phage genome will help advance the understanding of phage-host interaction patterns and phage physiology. This understanding could further facilitate the redesign of more robust phage genomes, paving the way for fundamental biological discoveries.

In order to solve these challenges, the embodiment of the present application provides a top-down method for continuous high-throughput deletion of genes to simplify the genome, that is, iteratively simplified phage genome method (CiPGr) relying on CRISPR-cas9. In the embodiments of the present application, the phage may be a tailed phage, or may be a tailless phage.

In order to illustrate the technical solutions provided by the present application, detailed descriptions will be given below in conjunction with specific drawings and embodiments.

Specifically, referring to Fig. 1, the method includes the following steps:

S10. Design and synthesize multiple CiPGr sequence libraries of two or more phages, and assemble each CiPGr sequence library with the pTarget plasmid backbone to form a CiPGr plasmid library.

In this step, the CiPGr sequence library of the phage is designed for the potentially deletable gene sequence. In the embodiment of the present application, the CiPGr sequence library can be designed for two or more phages at the same time, wherein a CiPGr sequence library can be designed for one type of phage , it is also possible to design two CiPGr sequence libraries.

Each CiPGr sequence library includes n CiPGr sequences, the value of n depends on the number of potentially deletable genes in a phage, and n is a natural number greater than or equal to 2. In the embodiment of the present application, the CiPGr sequence at least includes guide RNA (guide RNA, gRNA), and the gRNA of each CiPGr sequence is different, so as to match different deletable genes in a phage. It should be understood that the known essential genes in the phage, or the homologous essential genes of the phage on the US National Center for Biotechnology Information (NCBI), do not need to design guide RNA (guide RNA, gRNA).

In some embodiments, the CiPGr sequence comprises a barcode, two homology arms, a promoter, gRNA and primers. Among them, the barcode is used to distinguish different phage libraries, and different phages are separated by barcode primer PCR; the primer is a part of the gRNA sequence, which is used as the Gibson assembly site when "CiPGr sequence and pTarget plasmid backbone assembly" is performed for Gibson assembly .

In the examples of this application, all CiPGr sequences were synthesized on a DNA chip to realize batch synthesis of CiPGr sequence libraries. Exemplarily, all designed CiPGr sequence libraries were synthesized in a 6000-strip size DNA chip. In one embodiment, the length of the CiPGr sequence is designed to be 200 bp, so as to facilitate the synthesis of the gene sequence on the DNA chip and reduce the synthesis cost. Exemplarily, the CiPGr sequence is 200bp, wherein, the barcode is 20bp, the two homology arms are 50bp each, the promoter is 36bp, the spacer is 20bp, and the primer is 24bp.

In the embodiment of the present application, multiple CiPGr sequence libraries are split by PCR technology, that is, the CiPGr sequences in the CiPGr sequence library are separated by PCR according to different phages. Specifically, the CiPGr sequence library is separated by PCR through the primers and barcodes on the CiPGr sequence, and multiple CiPGr sequence libraries are split into a single CiPGr sequence library. Exemplarily, the cycle conditions for PCR separation are: 98°C, 2 minutes; 98°C, 10s, 58°C, 20s, 72°C, 6s; 72°C, 10 minutes, and stored at 4°C.

In the examples of this application, each CiPGr sequence library was assembled with the pTarget plasmid backbone, and the CiPGr sequence in the CiPGr sequence library was assembled with the pTarget plasmid backbone to form a CiPGr plasmid. A CiPGr plasmid library.

In some embodiments, the pTarget plasmid backbone includes Cm ^R and Ori, wherein Cm ^R represents the chloramphenicol resistance gene, and the origin of Ori restricted replication. In some embodiments, the pTarget plasmid backbone can be amplified by PCR, and the pTarget plasmid backbone obtained by PCR amplification can be purified by gel electrophoresis. In some embodiments, before the CiPGr sequence library is assembled with the pTarget plasmid backbone, the CiPGr sequence library is purified by gel electrophoresis.

In some embodiments, the method for assembling each CiPGr sequence library with the pTarget plasmid backbone is as follows: after connecting each CiPGr sequence library and the pTarget plasmid backbone through Gibson assembly reaction, and then purifying. Exemplarily, the ligation of each CiPGr library to the pTarget plasmid backbone was achieved through 4 parallel Gibson assembly reactions. Thus, a pTarget plasmid containing the CiPGr sequence is obtained, and different single host cells may contain different pTarget plasmids.

S20. Transform the CiPGr plasmid in the CiPGr plasmid library, screen transformed cells containing the CiPGr plasmid, and extract the CiPGr plasmid from the transformed cells; transform the CiPGr plasmid into a host bacterium containing the spCas9 plasmid to obtain a CiPGr plasmid-bacterial library , the CiPGr plasmid-bacteria library contains n kinds of CiPGr plasmid-bacteria.

In this step, the CiPGr plasmids in the CiPGr plasmid library are transformed to enrich the number of CiPGr plasmids. In some embodiments, the CiPGr plasmids in each CiPGr plasmid library are transformed into DH5α Escherichia coli competent cells, transformed 2 to 10 times in parallel, and the transformed DH5α Escherichia coli competent cells are collected; the DH5α Escherichia coli competent cells are plated Cultivate on a petri dish containing chloramphenicol and kanamycin, screen DH5α Escherichia coli transformed cells containing CiPGr plasmid, and extract CiPGr plasmid from DH5α Escherichia coli transformed cells.

Exemplarily, the way of culturing DH5α Escherichia coli competent cells on a petri dish containing chloramphenicol and kanamycin may be: culturing overnight at 37°C.

The CiPGr plasmid is transformed into a host bacterium containing the spCas9 plasmid to obtain a CiPGr plasmid-bacteria library, and the CiPGr plasmid-bacteria library contains n kinds of CiPGr plasmid-bacteria. At this point, the host bacterium contains a two-plasmid system, namely the pTarget plasmid and the spCas9 plasmid. Wherein, the host bacterium containing the spCas9 plasmid is determined according to the type of phage. Exemplarily, when the phage contains at least one of tailed phage T7, T4, seszw and selz, the host bacteria containing the spCas9 plasmid can be Escherichia coli (Mg1655) containing the spCas9 plasmid and Salmonella typhimurium containing the spCas9 plasmid.

In the embodiment of the present application, the obtained CiPGr plasmid-bacteria library can be stored at -80°C.

In some embodiments, to assess the coverage of the designed CiPGr sequence library in the CiPGr plasmid-bacterial library, PCR against the CiPGr sequence library was performed and sequenced by HiSeq 2500 (Illumina). After removing low-quality data from the obtained raw sequencing data, compare the sequencing data to the designed CiPGr library sequence, and determine the 100% consistent ratio of each CiPGr library sequence in the plasmid library.

S30. Cultivate the wild-type phage and the corresponding CiPGr plasmid-bacteria library in the culture medium, transfer the mutant phage product to a fresh CiPGr plasmid-bacteria library for further cultivation, and repeat iterative cultivation to generate a mutant phage library.

In this step, the CiPGr plasmid-bacteria library was taken, added to the culture medium, and cultivated overnight. When the CiPGr plasmid-bacteria library is a CiPGr plasmid-bacteria library stored at -80°C, after thawing the CiPGr plasmid-bacteria library on ice, take the CiPGr plasmid-bacteria library and add it to the culture medium. Exemplarily, the culture temperature is 37° C. Exemplarily, the medium can be LB medium. In some embodiments, in order to prevent other bacteria from contaminating the CiPGr plasmid-bacteria library, antibiotics and L-arabinose are added to the culture medium.

In one embodiment, CiPGr plasmid-bacteria were added to LB medium, and antibiotics and L-arabinose were added for overnight culture.

The wild-type phage and the corresponding CiPGr plasmid-bacterial library were cultured in the culture medium, the phage infected the corresponding CiPGr plasmid-bacterial library, and the phage DNA was injected into the cells of the corresponding CiPGr plasmid-bacterial library. The gRNA encoded by pTarget in the CiPGr plasmid-bacteria guides the Cas9 nuclease encoded by spCas9 to bind to the target gene on the wild-type phage and make the target gene double-strand break. Repair by homologous recombination (HR) induced by homologous sequences, leading to deletion or disruption of phage genes. If the gene is not essential for phage growth, the offspring of the mutant phage can amplify without the gene. Wild-type phages infect different CiPGr plasmid-bacteria in the CiPGr plasmid-bacteria library, resulting in the deletion of different genes, resulting in mutant phage products. In the examples of the present application, wild-type phage and the corresponding CiPGr plasmid-bacteria library were purified in the culture medium for the mutant phage products produced after the first culture to form the first-generation mutant phage library.

It should be understood that, in the examples of the present application, the corresponding CiPGr plasmid-bacteria library refers to the CiPGr plasmid-bacteria that can be infected by wild-type phage.

Take the mutant phage product and transfer it to a fresh CiPGr plasmid-bacteria library to continue culturing, so that the mutant phage product can be transferred to fresh bacteria containing double plasmids, which may infect bacteria containing different CiPGr plasmids and delete different genes. After repeated iterative culture, the mutant phage products are continuously transferred to new double-plasmid-containing bacteria, ie, CiPGr plasmid-bacteria, which will continuously accumulate gene deletions on the wild-type phage genome. At the same time, the mutant strains with a growth advantage can produce more offspring, and eventually occupy a dominant position in the mutant population and become the dominant strain. In some embodiments, the number of repetitions of repeated iterative culture is 300-600 times, which facilitates the easy detection of non-essential genes, quasi-essential genes and essential genes.

It should be understood that the fresh CiPGr plasmid-bacterial library referred to in the examples of the present application refers to a CiPGr plasmid-bacterial library that has not been co-cultured with wild-type phage, and is not necessarily a freshly configured CiPGr plasmid-bacterial library. However, it should be understood that for the same wild-type phage, the types of the CiPGr plasmid-bacterial library for repeated iterative cultivation, the CiPGr plasmid-bacterial library for repeated iterative cultivation, and the CiPGr plasmid-bacterial library for the first cultivation process are consistent .

In some embodiments, the generation steps of the mutant phage library are:

The wild-type phage and the corresponding CiPGr plasmid-bacterial library were cultivated in the culture medium to generate the first-generation mutant phage product, the first-generation mutant phage product was centrifuged, and the supernatant was taken to obtain the first-generation mutant phage library; The first-generation mutant phage library and fresh CiPGr plasmid-bacteria library medium were continuously cultivated to generate the second-generation mutant phage product, and the second-generation mutant phage product was centrifuged to obtain the supernatant to obtain the second-generation mutant phage library; The second-generation mutant phage library and fresh CiPGr plasmid-bacteria library were cultured in the culture medium to generate the third-generation mutant phage product, centrifuged the third-generation mutant phage product, and took the supernatant to obtain the third-generation mutant phage library; repeat In this iterative culture step, the number of transfers reaches 300-600 times, and each mutant phage library is collected.

In this case, the continuous transfer of mutant phage products to new double-plasmid-containing bacteria, CiPGr plasmid-bacteria, allowed the accumulation of gene deletions on the wild-type phage genome, resulting in the formation of mutant phages lacking different genes, various Mutant phages with gene deletions form a mutant phage library. The various deleted genes in the mutant phage library form a set of deleteable genes.

In the above method, each time a part of the mutant phage library obtained after cultivation is added to a fresh CiPGr plasmid-bacteria library, and the other part is stored under low temperature conditions for further analysis. In some embodiments, in the repeated iterative cultivation steps, the titer concentration of the mutant phage library is regularly determined; when the concentration of the obtained mutant phage is less than 10 ⁵ /ml, wild-type host cells are used to cultivate the mutant phage library. Due to subculture, the number of mutant phages decreases. In this case, the number or concentration of mutant phages can be expanded by culturing wild-type host cells. Exemplarily, every 10 transfers, a titration concentration of the mutant phage library is determined.

S40. Sequencing the mutant phage library obtained after a certain number of transfers, and confirming all the deletable genes in the phage.

In this step, the mutant phage library obtained after a certain number of transfers is sequenced to obtain the deletable gene information of the mutant phage. In this step, after the mutant phage library is obtained, all stored mutant phages can be sequenced, or the obtained mutant phages can be sequenced during the process of culturing the mutant phages.

In some embodiments, PCR is used to monitor the deletion of a phage gene. Exemplarily, one μL of mutant phage supernatant is used as a template for PCR, and Ex Taq DNA polymerase (Takara, RR01AM) is used to amplify the target gene through appropriate primers, and the PCR method is performed by agarose gel electrophoresis and Sanger Sequencing verified deletion of the gene.

In the examples of the present application, in order to monitor the efficiency of gene deletion, the DNA of the mutant phage library was extracted according to the manual of the phage DNA isolation kit (NORGEN, 46850), and DNA sequencing was performed using a HiSeq1500 sequencer. Exemplarily, the 20th, 30th, 40th and 50th transfer products of phage T7 and T4 are sequenced, and then every 50 to 100 transfers are sequenced.

In a possible implementation, the method provided in the examples of the present application can also screen a single active mutant phage. In some embodiments, screening for a single active mutant phage includes:

Cultivate 1×10 ³ -5×10 ³ PFU mutant phage library and wild-type host cells in solid medium, randomly select several large plaques and small plaques for isolation and purification; host cells cultured overnight and fresh LB The culture medium is added to a 96-well plate, and after adding a single mutant phage, it is cultured on a microplate reader, and the OD ₆₀₀ is detected every once in a while for 12 to 24 hours; the mutant phage is selected according to the bactericidal curve, and the bacteriophage is selected by the drawing method. The selected mutant phages were further purified.

Exemplarily, taking tailed phages T7, T4, seszw and selz as examples, each phage has two libraries (8 CiPGr plasmid-bacteria libraries are formed in total). To obtain a single mutant phage, 1×10 ³ PFU of the mutant phage library was mixed with 300 μL of wild-type host cells and 10 mL of 0.7% LB agar in a tube, poured into a plate, and incubated at 37 °C. overnight culture under conditions. Randomly pick 8 large and 8 small plaques, and then separate and purify. To determine the bactericidal effect of a single mutant phage, add 10 μL of overnight cultured host cells, 200 μL of fresh LB medium into a 96-well plate, and purify and pick a single phage plaque into a 96-well plate, and run on a microplate reader The culture was carried out at 37°C, and the OD600 was detected every 10 minutes for 12 hours; the mutant phages were selected according to the bactericidal curve, that is, the one-step growth curve of the phage, and further purified by the plating method to obtain a single mutant phage with activity.

Exemplarily, the one-step growth curve determination method of phage is: after bacterial streaking, pick a single clone for overnight culture; freshly culture phage, measure the concentration, and dilute to 10 ⁵ PFU for later use; preheat the medium, inoculate 10ml with 1% volume, A total of 3 tubes were cultured at 37°C for 2 hours, centrifuged at 7000g at room temperature, removed the supernatant, added 5ml of preheated medium, oscillated and mixed; added 0.2mmol/L CaCl, added 100μL phage, oscillated and mixed; stood at room temperature for 5 minutes Finally, add 30ml of preheated culture medium, shake and mix well; after cultivating for a certain period of time, take samples and continue to detect the concentration.

In the embodiment of the present application, through the above-mentioned steps, a simplified phage genome with activity can be obtained quickly and with high throughput, especially for new phages with a large number of unknown functional genes.

In the process of iteratively culturing the wild-type phage and the corresponding CiPGr plasmid-bacteria library, gene deletions on the wild-type phage genome continue to accumulate, and at the same time, the phage mutants with growth advantages can produce more offspring, And eventually occupy a dominant position in the mutant population and become the dominant strain.

In the examples of this application, the phage mutants were isolated and single-clonal sequenced to obtain a simplified phage genome; and the phage mutant libraries were mixed together for competitive culture, and mutants with stronger properties than wild-type phages could be isolated. In a possible implementation, the method provided in the examples of the present application can also screen dominant mutant phages, and the screening steps include:

After mixing the mutant phages in the mutant phage library, co-culture with the corresponding wild-type host cells, transfer to fresh wild-type host cells for re-cultivation, and repeat the steps of "transfer to fresh wild-type host cells for re-culture" N-1 times, get TN mutant phage ((wherein, T represents transfer, N represents transfer number));

Mix 1×10 ³ -5×10 ³ PFU of TN mutant phage, wild-type host cells and LB agar, pour into LB plates, and culture overnight;

After selecting different plaques for purification, overnight cultured host cells and fresh LB medium were added to a 96-well plate, and a single plaque was purified and transferred to a 96-well plate, cultured on a microplate reader, and Test the OD ₆₀₀ at intervals for 12-24 hours; screen the dominant mutant phages according to the bactericidal curve.

Exemplarily, taking the tailed phages T7, T4, seszw and selz as an example, the mutant phage libraries of different transfers were mixed together, and the mixed mutant phage libraries (10 ⁵ PFU) and the corresponding logarithmic growth (10 ⁸ PFU ) wild-type host cells were co-cultured for 2 hours to obtain T1 mutant phages; part of the T1 mutant phages were transferred to fresh host cells for culture again, so that a total of 8 transfers were performed, and a total of 16-20 generations were amplified to obtain T8 mutant phages. 10 ³ PFU of T8 mutant phage, 300 μL of wild-type host cells and 10 mL of 0.7% LB agar were added to a tube, mixed, poured into LB plates, and incubated overnight at 37°C. Pick 3 different plaques for purification. To determine the bactericidal effect of a single mutant phage, add 10 μL of overnight cultured host cells, 200 μL of fresh LB medium to a 96-well plate, and purify and transfer a single plaque to a 96-well plate, on a microplate reader, Culture was carried out at 37°C, and OD600 was detected every 10 minutes for 12 hours.

In the embodiment of the present application, non-essential genes, quasi-essential genes and essential genes can be easily detected by performing metagenomic sequencing on the mutant phage library. In a possible implementation, the method provided in the examples of the present application further includes the determination of non-essential genes, quasi-essential genes and essential genes in the phage.

In some embodiments, the determination of non-essential genes, quasi-essential genes and essential genes in phages includes:

Analyze the probability of deletion genes of mutant phages in the mutant phage library. Deletable genes with a deletion frequency of <5% are determined to be quasi-essential genes of the phage, and deletable genes with a deletion frequency of >5% are determined to be non-essential genes of the phage. Deletable genes Genes for which no deletions were detected were identified as essential genes for the phage.

It should be understood that the frequency of a gene mutation is expressed as a percentage of gene deletion/gene retention in a mutant library. During the iterative culture of the wild-type phage and the corresponding CiPGr plasmid-bacteria library, the gene deletions on the genome of the wild-type phage continued to accumulate. According to the results of the iterative culture, the phage genes could be initially classified into three groups: (1 ) genes deleted in isolated single mutant phage genomes, with a relatively high frequency of gene deletions of more than 5%, were classified as non-essential genes. (2) Other genes whose frequency of gene deletion in the deleteable gene set was <5% were classified as quasi-essential genes. Deletion of these genes produces defective phage with a worse growth advantage than mutants that delete non-essential genes. (3) Genes not detected in the deleteable gene set are important for phage growth and classified as essential genes.

Exemplarily, taking tailed bacteriophages T7, T4, seszw and selz as examples, in isolated single mutant phage genomes, most of the detected gene deletions (T7 100%, T4 92.4%, seszw 94.1%, selz 98.4%) showed a relatively high frequency > 5% in the transferred mutant phage library, which indicated that these genes were less important to phage growth than other genes (frequency < 5%).

In the embodiment of the present application, the minimum phage genome and the phage with stronger bactericidal ability can be obtained by identifying the essential genes, non-essential genes and quasi-essential genes of the phage.

It should be understood that eukaryotic viruses can also simplify the genome by deleting genes through high-throughput deletion according to the methods provided in the examples of the present application. In the above method, the phage is correspondingly replaced by eukaryotic virus, the host bacterium is correspondingly replaced by host cell, and the CiPGr plasmid-bacteria is correspondingly replaced by CiPGr plasmid-cell.

Referring to Fig. 2 below, taking tailed phages T7, T4, seszw and selz as examples, the phage genome simplification method, the screening and determination of non-essential genes and quasi-essential genes in phage genes, and the identification of dominant strains will be described in conjunction with specific examples. Form and screen.

(1) CiPGr plasmid library design, construction and transformation

A total of 8 CiPGr sequence libraries of 200 bp were designed for the potential deleteable gene sequences of tailed phages T7, T4, seszw and selz, 2 CiPGr sequence libraries for each phage. Among them, the CiPGr sequence is shown in Figure 2-a, including barcode (20bp), homology arm (50×2bp), promoter (36bp), spacer (20bp) and primer (24bp).

All designed CiPGr libraries were synthesized on a 6000-strip size DNA chip as shown in Fig. 2-c-1. The cassette-100 (disrupted gene) and cassette-gene cassettes (deleted gene) of the four phages were separated by PCR, and the cycle conditions for PCR separation were: 98°C, 2 minutes; 98°C, 10s, 58°C, 20s, 72 ℃, 6s; 72℃, 10 minutes, and store at 4℃.

As shown in Figure 2-c-2, the pTarget plasmid target backbone was amplified by PCR, and the plasmid backbone and CiPGr library were purified by gel electrophoresis. Purification was performed after assembling each CiPGr sequence library with the pTarget plasmid backbone by 4 parallel Gibson assemblies to form a CiPGr plasmid library. Transform the purified CiPGr plasmid library into DH5α Escherichia coli competent cells, transform each library 2 to 10 times in parallel, and collect the transformed DH5α Escherichia coli competent cells; spread the DH5α Escherichia coli competent cells in a 15cm ² culture dish , add chloramphenicol and kanamycin, and culture overnight at 37°C. CiPGr plasmids were extracted from DH5α E. coli transformed cells after the colonies were scraped off the dishes. Eight CiPGr plasmid libraries were transformed into Escherichia coli (Mg1655, containing the spCas9 plasmid) and Salmonella typhimurium (containing the spCas9 plasmid), to obtain the CiPGr plasmid-bacterial library, which was stored at -80°C for future use.

In this step, in order to evaluate the coverage of the CiPGr library designed in the plasmid library, PCR was performed on the CiPGr library and sequenced by HiSeq 2500 (Illumina). The low-quality data was removed from the obtained raw sequencing data, and the sequencing data was compared to the designed CiPGr library sequence to determine the 100% consistent ratio of each CiPGr library sequence in the plasmid library.

(2) Generation of mutant phage library and metagenomic sequencing

Take out the CiPGr-containing plasmid-bacterial library stored at -80°C. After melting on ice, take out 300 μL of each library, add it to 15ml LB medium, add antibiotics and L-arabinose at the same time, and culture overnight at 37°C. As shown in Figure 2-c-3, add 1ml of phage (10 ⁹ PFU/ml), 1ml of the corresponding host cell containing the plasmid library (10 ⁹ CFU/ml) and 1ml of LB medium into the shaker tube, and incubate at 37°C for 6 hours. Generation of first generation mutant phage products. Take 1mL of the above-mentioned first-generation mutant phage product, centrifuge to get the supernatant, and transfer it to 1ml of fresh CiPGr plasmid-bacteria library and 1ml LB medium for culture; as the above process, the transfer is carried out 300-600 times. Figure 3 provides a library of phage-deletable genes, showing the frequency of deletion of various genes in the mutant library of phages transferred at the nth transfer. The deletable gene library is produced by transfer of two types of plasmid libraries (gene destruction library, gene deletion library), where the frequency indicates the percentage (log2) of the corresponding reads (deletion or destruction) of the gene in the population, and the corresponding heat Figures were generated by the phatmap R package. "L" indicates deletion of a large segment that we did not design; "P" indicates deletion of one or two bases in the coding region, resulting in premature codon termination.

Among them, 1 ml of the supernatant generated in each transfer was used for subculture, and 1 ml was stored at 4° C. for further analysis. Every 10 transfers, a titration concentration of the mutant phage library was determined. If the mutant phage concentration is less than 10 ⁵ /ml, wild-type host cells are used to grow the mutant phage library.

Deletion of phage genes was monitored by PCR every 10 transfers. 1 μL of the supernatant was used as a template for PCR using Ex Taq DNA polymerase (Takara, RR01AM), and amplification was performed on the target gene with appropriate primers. Gene deletions were verified by agarose gel electrophoresis and Sanger sequencing. As shown in Figure 4, PCR monitoring of phage gene deletion is provided, and the deletion efficiency of gp4.7 and gp5.3 in the phage T7 genome is detected by PCR and gel electrophoresis. In the figure, the band sizes of gp4.7 and gp5.3 were reduced from 559bp and 455bp to 128bp and 117bp, respectively.

In order to monitor the efficiency of gene deletion, the DNA of the mutant phage library was extracted according to the manual of the phage DNA isolation kit (NORGEN, 46850), and the DNA was sequenced with a HiSeq1500 sequencer. We sequenced the 20th, 30th, 40th, and 50th transfer products of phages T7 and T4, and then every 50 to 100 transfers thereafter.

(3) Screening, characterization and genome sequencing of single isolated mutant phages

As shown in Figure 2-c-4, in order to obtain a single mutant phage, 10 ³ PFU of the mutant phage library was mixed with 300 μL of wild-type host cells and 10 mL of 0.7% LB agar in a tube, and poured Plates were incubated overnight at 37°C. Randomly pick 8 large and 8 small plaques, and then separate and purify.

To determine the bactericidal effect of a single mutant phage, 10 μL of overnight cultured host cells, 200 μL of fresh LB medium were added to a 96-well plate, and a single phage plaque was purified and picked into a 96-well plate. Incubate on a microplate reader at 37°C, and detect OD600 every 10 minutes for 12 hours; select mutant phages according to the bactericidal curve, that is, the one-step growth curve of phages, and further purify them by drawing the plate method. Among them, the method for determining the one-step growth curve of bacteriophage is as follows: after bacterial streaking, pick a single clone for overnight culture; freshly culture phage, measure the concentration, and dilute to 10 ⁵ PFU for later use; 3 tubes, after incubating at 37°C for 2 hours, centrifuge at 7000g at room temperature, remove the supernatant, add 5ml of preheated medium, shake and mix well; add 0.2mmol/L CaCl, add 100μL phage, shake and mix well; after standing at room temperature for 5min , add 30ml of preheated culture medium, vortex and mix well; after culturing for a certain period of time, take samples and continue to detect the concentration.

Fig. 5 is the phage plaque of mutant phage T7 in the double-layer agar plate; Fig. 6 is the bactericidal curve of mutant phage T7 and T4 to MG1655, and the bactericidal curve of mutant phage seszw and selz to Salmonella ST56.

(4) Screening, characterization and genome sequencing of stronger mutant phages

Mix the mutant phage libraries of different transfers together, mix the mutant phage libraries (10 ⁵ PFU) with the corresponding logarithmic growth (10 ⁸ PFU) wild-type host cells Co-cultured for 2 hours to obtain T1 mutant phages; part of the T1 mutant phages were transferred to fresh host cells for culture again, so that a total of 8 transfers were performed, and a total of 16-20 generations were amplified to obtain T8 mutant phages. 10 ³ PFU of T8 mutant phage, 300 μL of wild-type host cells and 10 mL of 0.7% LB agar were added to a tube, mixed, poured into LB plates, and incubated overnight at 37°C. Pick 3 different plaques for purification. To determine the bactericidal effect of a single mutant phage, add 10 μL of overnight cultured host cells, 200 μL of fresh LB medium to a 96-well plate, and purify and transfer a single plaque to a 96-well plate, on a microplate reader, Culture was carried out at 37°C, and OD600 was detected every 10 minutes for 12 hours.

Fig. 7 is the bactericidal curve of the stronger phage of screening, wherein, a is the bactericidal curve of stronger T7 and T4 mutant strain to MG1655 and stronger seszw and selz mutant strain to Salmonella (ST56); b is T7, seszw, T4 and One-step growth curves of stronger phages from Selz. In the figure, WT represents the wild-type phage; the data are expressed as the mean ± SD of three experiments; the phage titer was determined by the PFU method.

Fig. 8 is the gene map of the stronger phage screened, wherein the black bars indicate gene deletion regions; the black dots indicate point mutations. Arrows indicate genes, and the direction of the arrow corresponds to the direction of transcription and translation.

(5) Data Analysis

The frequency of a gene mutation is expressed as the percentage of gene deletion/gene retention in a mutant library. We used the open source software breseq (version 0.28.0) to predict point mutations and gene deletions for each mutant library; the genome assembly of mutant phages used SOAP-denove (V2.04-r241) software.

Application of CiPGr to four different tailed phages (model phages T7 and T4; Salmonella phages seszw and selz) resulted in mutants of these phages deleting 8%–23% (3.3–35kbp) of their sequences, resulting in simplified phages skeleton. Macro-set sequencing of mutant phage libraries showed that non-essential and quasi-essential genes accounted for 46.7% to 65.4% of the total number of these phage genes. Loss of quasi-essential genes (24%–26%) can severely impair phage amplification, causing the corresponding mutants to gradually disappear in the mutant library, resulting in no detection of quasi-essential genes in the genomes of isolated single mutants. delete.

Mutant phages with stronger bactericidal ability than wild-type phages were obtained through screening. We observed that the stronger mutants screened performed faster than their wild-type counterparts in killing the host (5 minutes faster for T7, 2 hours faster for T4, 1 hour faster for seszw, and 2 hours faster for selz).

The above results demonstrate that CiPGr is a general and efficient method applicable to novel phages and other eukaryotic viruses without any prior knowledge.

The above are only optional embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, various modifications and changes may occur in this application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included within the scope of the claims of the present application.

Claims (9)

1. A method for simplifying phage genome frameworks in high throughput is characterized by comprising the following steps:

designing and synthesizing a plurality of CiPGr sequence libraries of two or more than two phages, and respectively assembling each CiPGr sequence library with a pTarget plasmid skeleton to form a CiPGr plasmid library; wherein the CiPGr sequence library comprises n CiPGr sequences, n is a natural number greater than or equal to 2, the CiPGr sequences comprise gRNAs, and the gRNAs of each CiPGr sequence are different;

transforming CiPGr plasmids in the CiPGr plasmid library, screening transformed cells containing the CiPGr plasmids, and extracting the CiPGr plasmids from the transformed cells; transforming the CiPGr plasmid into host bacteria containing spCas9 plasmids to obtain a CiPGr plasmid-bacteria library, wherein the CiPGr plasmid-bacteria library contains n CiPGr plasmid-bacteria;

culturing wild phage and the corresponding CiPGr plasmid-bacterium library in a culture medium, transferring a mutant phage product to a fresh CiPGr plasmid-bacterium library for continuous culture, and repeating iterative culture to generate a mutant phage library;

sequencing the mutant phage library obtained after a certain transfer times to confirm all the deletable genes in the phage.

2. The method for high throughput simplification of phage genome framework according to claim 1, characterized in that the generation step of the mutant phage library is:

culturing the wild phage and the corresponding CiPGr plasmid-bacterium library in a culture medium to generate a first generation mutant phage product, centrifuging the first generation mutant phage product, and taking supernatant to obtain a first generation mutant phage library; continuously culturing part of the first generation mutant phage library and a fresh CiPGr plasmid-bacteria library culture medium to generate a second generation mutant phage product, centrifuging the second generation mutant phage product, and taking supernatant to obtain a second generation mutant phage library; culturing part of the second generation mutant phage library and the fresh CiPGr plasmid-bacterium library in a culture medium to generate a third generation mutant phage product, centrifuging the third generation mutant phage product, and taking supernatant to obtain a third generation mutant phage library; repeating the iterative culture step to ensure that the transfer times reach 300-600 times, and collecting each mutant phage library.

3. The method for high throughput simplification of a phage genome backbone according to claim 1, characterized in that the method further comprises screening a single active mutant phage.

4. The method for high throughput simplification of phage genome backbone of claim 3, wherein said screening individual active mutant phage comprises:

will be 1 × 10 ³ ～5×10 ³ Culturing a mutant phage library of the PFU and wild host cells in a solid culture medium, and randomly selecting a plurality of large bacterial plaques and small bacterial plaques for separation and purification; adding overnight cultured host cells and fresh LB culture medium into 96-well plate, adding single mutant phage, culturing on enzyme labeling instrument, and detecting OD once every a period of time ₆₀₀ Lasting for 12 to 24 hours; mutant phages were selected according to the bactericidal curve and the selected mutant phages were further purified by the scratching method.

5. The method for high throughput simplification of a phage genome backbone according to any of claims 1 to 4, characterized in that the method further comprises screening for dominant mutant phages, comprising the steps of:

mixing the mutant phages in the mutant phage library, co-culturing with corresponding wild-type host cells, transferring to fresh wild-type host cells for re-culture, and repeating the step of transferring to fresh wild-type host cells for re-culture for N-1 times to obtain TN mutant phages;

will be 1 × 10 ³ ～5×10 ³ Mixing TN mutant phage of PFU, wild host cell and LB agar, pouring into LB plate, culturing overnight;

selecting different plaques for purification, adding overnight cultured host cells and fresh LB culture medium into a 96-well plate, purifying and transferring single plaques into the 96-well plate, culturing on a microplate reader, and culturing at intervalsIndirect detection of one-time OD ₆₀₀ Lasting for 12 to 24 hours; and (4) screening dominant mutant phages according to the bactericidal curve.

6. The method for high throughput simplification of a bacteriophage genomic backbone according to any one of claims 1 to 4, wherein said method further comprises determination of non-essential genes, quasi-essential genes and essential genes in said bacteriophage.

7. The method for high throughput simplification of phage genome backbone according to claim 6, wherein the determination of non-essential genes, quasi-essential genes and essential genes in the phage comprises:

analyzing the probability of the deletion genes of the mutant bacteriophage in the mutant bacteriophage library, wherein the deletable genes with the deletion frequency of less than 5 percent are determined as quasi-essential genes of the bacteriophage, the deletable genes with the deletion frequency of more than 5 percent are determined as non-essential genes of the bacteriophage, and the genes of which the deletion is not detected are determined as essential genes of the bacteriophage.

8. The method for high throughput simplification of a phage genome backbone of any one of claims 1 to 4, wherein after transformation of the CiPGr plasmid in the CiPGr plasmid library, screening transformed cells containing the CiPGr plasmid and extracting the CiPGr plasmid from the transformed cells comprises:

transforming the CiPGr plasmid in the CiPGr plasmid library into DH5 alpha escherichia coli competent cells, parallelly transforming for 2-10 times, and collecting the transformed DH5 alpha escherichia coli competent cells; and (3) paving the DH5 alpha escherichia coli competent cells on a culture dish containing chloramphenicol and kanamycin for culture, screening DH5 alpha escherichia coli transformed cells containing the CiPGr plasmid, and extracting the CiPGr plasmid from the DH5 alpha escherichia coli transformed cells.

9. The method for high throughput simplification of a phage genome backbone according to any one of claims 1 to 4, wherein the method for assembling the CiPGr sequence library with pTarget plasmid backbone separately is: and (3) connecting the CiPGr sequence library and the pTarget plasmid skeleton through a Gibson assembly reaction, and purifying.

Images