CN114934059B - Method for simplifying phage genome framework in high flux - Google Patents
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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
Technical Field
The application relates to the technical field of life science, in particular to a method for simplifying a bacteriophage genome framework in a high-throughput manner.
Background
Bacteriophages are the most abundant and genetically diverse organisms on earth. For over a century, phage research has been the key to many biological discoveries, providing an important biotechnological tool for molecular biology. In recent years, viral metagenomic sequencing has shown that a large number of phage sequences are found in the human gut and other environments. Most (> 75%) of these sequences are novel, with more than 95% of the sequences belonging to the tailed double stranded (ds) DNA phage. In addition, bacteriophages have been identified as potential natural antibacterial agents for the treatment of bacterial infections. However, the phage has problems of host specificity, easy generation of resistance and the like as a potential natural antibacterial agent for treating bacterial infection, and the pharmaceutical property of the phage is directly influenced. Therefore, researchers have made many synthetic biological efforts on bacteriophages to overcome these limitations.
Tailless phages such as M13 and X174, have very compact and small genomes (< 10 kb), making it easy to edit and understand the function of their genes, since they encode a limited number of genes that are relatively simple. The genome of the tailed phage is usually relatively large in number (14-500 kbp) and is abnormally diversified, which makes it challenging to obtain the simplified genome of the tailed phage on a large scale. One of the challenges is: within genome-scale, there is no efficient way to identify nonessential genes of bacteriophages. For example, 25 nonessential genes of the model phage T7 were obtained from the accumulated knowledge from a large number of studies in the last thirty-four decades. The amplification of bacteriophages depends entirely on their host, and their unique self-propagating properties, make the methods widely used in bacteria (simplified genomic methods widely used in microorganisms such as E.coli, yeast, bacillus and Mycoplasma, such as homologous recombination, tn5 mutation, etc.) potentially unsuitable for large-scale application in tailed bacteriophages. In addition, due to the highly diverse nature of the phage genome, it is difficult to obtain nonessential genetic information by bioinformatics approach alignment.
The de novo synthesis of simplified genomes requires high throughput identification of nonessential genes of phages, but high throughput identification methods such as the dCas9 method fail for two reasons: firstly, because the phage genome can be replicated in a plurality of copies in bacteria, the gene expression of a plurality of phage genomes can not be completely inhibited by dCas9, and a false positive result is caused; second, many of the phage genes are transcribed in tandem, and inhibition of the upstream gene by dCas9 will result in the suppressed expression of all downstream genes, leading to false negative results. The Tn 5-like transposon method also results in a reduction in the efficiency of identification due to the unique growth pattern of the phage.
Disclosure of Invention
The application aims to provide a method for simplifying a bacteriophage genome framework with high flux, and aims to solve the problems of complexity and low efficiency of the method for simplifying the bacteriophage genome.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
a method for simplifying phage genome framework with high flux comprises 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 the CiPGr sequences 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 plasmid 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.
In one embodiment, the step of generating the library of mutant phages is:
culturing the wild phage and the corresponding CiPGr plasmid-bacteria 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-bacteria 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 make the transfer times reach 300-600 times, and collecting all the mutant phage libraries.
In one embodiment, the method further comprises screening a single active mutant phage.
In one embodiment, the screening of a single 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.
In one embodiment, the method further comprises screening for dominant mutant phages, comprising the steps of:
mixing the mutant phages in the mutant phage library, co-culturing the mutant phages with wild host cells, transferring the mutant phages to fresh wild host cells for re-culture, and repeating the step of transferring the mutant phages to fresh wild 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 a single plaque into the 96-well plate, culturing on a microplate reader, and detecting OD once every other period 600 Lasting for 12 to 24 hours; and (4) screening dominant mutant phages according to the bactericidal curve.
In one embodiment, the method further comprises the determination of non-essential, quasi-essential, and essential genes in the bacteriophage.
In one embodiment, the determination of non-essential, quasi-essential, and essential genes in the bacteriophage comprises:
analyzing the probability of the deletion gene of the mutant bacteriophage in the mutant bacteriophage library, wherein the deletable gene with the deletion frequency of less than 5 percent is determined as the quasi-essential gene of the bacteriophage, the deletable gene with the deletion frequency of more than 5 percent is determined as the non-essential gene of the bacteriophage, and the gene without deletion detection is determined as the essential gene of the bacteriophage.
In one embodiment, the CiPGr sequence further comprises a barcode, two homology arms, a promoter and a primer.
In one embodiment, after transformation of a CiPGr plasmid from said library of CiPGr plasmids, selection of transformed cells containing said CiPGr plasmid, extraction of said CiPGr plasmid from said 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.
In one embodiment, the step of assembling all the CiPGr sequences in the CiPGr sequence library with pTarget plasmid backbone separately is preceded by PCR isolation of the CiPGr sequences in the CiPGr sequence library from different phage.
In one embodiment, the cycling conditions for the PCR separation are:
2 minutes at 98 ℃;98 ℃,10s,58 ℃,20s,72 ℃ for 6s; the samples were stored at 72 ℃ for 10 minutes and 4 ℃.
In one example, the method for assembling the CiPGr sequence libraries separately to pTarget plasmid backbone is: and (3) connecting the CiPGr sequence library and the pTarget plasmid skeleton through a Gibson assembly reaction, and purifying.
In one embodiment, the step of culturing wild-type phage and said corresponding CiPGr plasmid-bacteria library in culture medium is preceded by the step of: the CiPGr plasmid-bacteria library was added to LB medium and cultured overnight with antibiotics and L-arabinose.
In one embodiment, the number of repetitions of the iterative culture is 300 to 600.
In one embodiment, in the step of repeating the iterative culturing, the titer concentration of the mutant phage library is periodically determined; when the concentration of the obtained mutant phage is less than 10 5 At individual/ml, the mutant phage library was cultured using wild-type host cells.
In one embodiment, the phage is a tailed phage, tailless phage, or other eukaryotic virus.
In one embodiment, the phage replacement is a eukaryotic virus, the host bacterial replacement is a host cell, and the CiPGr plasmid-bacterium replacement is a CiPGr plasmid-cell.
The method for simplifying the phage genome framework in high throughput provided by the embodiment of the application has the beneficial effects that: aiming at a deletable gene of a phage, a CiPGr sequence library with different gRNAs in the CiPGr sequence is designed, the CiPGr sequence and a pTarget plasmid framework are assembled to form a CiPGr plasmid (namely pTarget plasmid), and the CiPGr plasmid is transformed into host bacteria containing spCas9 plasmid to obtain a CiPGr plasmid-bacteria library. At this time, the host bacteria contain both pTarget plasmid and spCas9 plasmid, and different individual bacteria in the CiPGr plasmid-bacterial library may contain different pTarget plasmids. Infecting a CiPGr plasmid-bacterium with a wild type phage, guiding RNA (gRNA) encoded by pTarget to guide the binding of a SPCas9 encoded Cas9 nuclease to a target gene, and enabling the target gene to be subjected to double strand break, and repairing through Homologous Recombination (HR) induced by a homologous sequence to cause gene deletion or damage. If the gene is not essential for phage growth, progeny of the mutant phage can be amplified without the gene. Through iterative culture, the phage infects different CiPGr plasmid-bacteria, resulting in the deletion of different genes, thereby continuously producing mutant phage. The mutated phages are continuously transferred to a new two-plasmid containing bacterium, i.e.CiPGr plasmid-bacterium, which may infect host bacteria containing different pTarget plasmids and delete different genes. And (3) continuously deleting genes at high flux from top to bottom, continuously deleting and accumulating the genes on the phage genome to obtain a mutant phage library containing different deleted genes, and sequencing the mutant phage obtained after a certain transfer times until a phage genome framework is obtained. The method can rapidly achieve high-throughput simplification of the genome backbone of all phages, including tailed and tailless phages.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the embodiments or the prior art description will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings may be obtained according to these drawings without inventive labor.
FIG. 1 is a schematic flow chart of a method for high throughput simplification of phage genome backbone provided in the examples herein;
FIG. 2 is a flow chart of a method for high throughput simplification of phage genome backbone provided in the examples of this application;
FIG. 3 is a library of phage-deletable genes provided in example 1 of the present application;
FIG. 4 is a gel electrophoresis of gp4.7 and gp5.3 from bacteriophage T7 genome in different numbers of transfers in the mutants provided in example 1 of the present application;
FIG. 5 is a plaque of mutant phage T7 in a double-layered agar plate as provided in example 1 of the present application;
FIG. 6 is a graph of the bactericidal effect of mutant bacteriophages T7 and T4 on MG1655 provided in example 1 of the present application;
FIG. 7 is a bactericidal curve for phage with greater bacteriostatic capacity than wild-type, as provided in example 1 of the present application;
FIG. 8 is a genetic map of a more strongly mutated bacteriophage provided in example 1 of the present application.
Detailed Description
In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
The terms "first", "second" and "first" are used merely for descriptive purposes and are not to be construed as indicating or implying relative importance or to implicitly indicate a number of technical features. The meaning of "plurality" is two or more unless specifically limited otherwise.
Phage synthetic biology can integrate functional genes or gene lines into the phage genome, enhancing their antibacterial activity and potential in phage therapy and other diverse bioengineering applications. Such genetic line integration can be challenging because of the limited DNA packaging space of the phage particle. The accessory genes of the phage may help the phage to better adapt to a wide range of ecoenvironments, but it may not be necessary under given conditions. 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 to facilitate understanding of the phage-host interaction pattern and phage physiology. This understanding can further facilitate the redesign of more powerful phage genomes, paving the way for basic biological discovery.
To address these challenges, the present embodiments provide a top-down method of continuous high-throughput gene deletion to simplify genomes, i.e., an iterative simplified phage genome approach (CiPGr) that relies on CRISPR-cas 9. In the embodiment of the application, the phage can be tailed phage or tailless phage.
In order to explain the technical solutions provided in the present application, the following detailed description is made with reference to specific drawings and examples.
Specifically, referring to fig. 1, the method includes the following steps:
s10, designing and synthesizing a plurality of CiPGr sequence libraries of two or more phages, and assembling each CiPGr sequence library with a pTarget plasmid skeleton to form a CiPGr plasmid library.
In the step, a CiPGr sequence library of the phage is designed aiming at potential deletable gene sequences, and the embodiment of the application can simultaneously design the CiPGr sequence library for two or more phage, wherein one phage can design one CiPGr sequence library and two CiPGr sequence libraries can be designed.
Each CiPGr sequence library comprises n CiPGr sequences, the value of n depends on the number of potential deletable genes in a phage, and n is a natural number greater than or equal to 2. In the examples of this application, the CiPGr sequences include at least guide RNAs (gRNAs), and the gRNAs for each CiPGr sequence are different to match different deletable genes in a single phage. It is understood that essential genes known in phage, or homologous essential genes of phage at the National Center for Biotechnology Information (NCBI) in the united states, do not require the design of guide RNAs (grnas).
In some embodiments, the CiPGr sequence comprises a barcode, two homology arms, a promoter, a gRNA, and a primer. Wherein, the bar code is used for distinguishing different phage libraries, and different phage are separated by the bar code primer PCR; the primer is a part of the gRNA sequence, and serves as a site for Gibson assembly when Gibson assembly is performed by assembling the "CiPGr sequence to pTarget plasmid backbone".
In the embodiment of the application, all CiPGr sequences are synthesized on a DNA chip, so that batch synthesis of a CiPGr sequence library is realized. Illustratively, all libraries of designed CiPGr sequences were synthesized in a 6000-size DNA chip. In one embodiment, the CiPGr sequence is designed to be 200bp in length to facilitate synthesis of gene sequences on DNA chips and reduce synthesis cost. Illustratively, the CiPGr sequence is 200bp, where 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 application, a plurality of CiPGr sequence libraries are split by a PCR technology, namely, the CiPGr sequences in the CiPGr sequence libraries are subjected to PCR separation according to different phages. Specifically, a CiPGr sequence library is subjected to PCR separation through primers and barcodes on the CiPGr sequence, and a plurality of CiPGr sequence libraries are split into a single CiPGr sequence library. Exemplary cycling conditions for PCR isolation are: 2 minutes at 98 ℃;98 ℃,10s,58 ℃,20s,72 ℃,6s; the samples were stored at 72 ℃ for 10 minutes and 4 ℃.
In the embodiment of the application, each CiPGr sequence library is assembled with a pTarget plasmid framework, the CiPGr sequences in the CiPGr sequence library are assembled with the pTarget plasmid framework to form a CiPGr plasmid, and the CiPGr plasmids formed after the CiPGr sequences in the CiPGr sequence library are assembled are combined to form a CiPGr plasmid library.
In some embodiments, the pTarget plasmid backbone comprises Cm R And Ori, wherein Cm R Indicating the origin of chloramphenicol resistance gene, ori limited 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, the CiPGr library is purified by gel electrophoresis prior to assembly with the pTarget plasmid backbone.
In some embodiments, the method for assembling each CiPGr sequence library separately to the pTarget plasmid backbone is: each CiPGr sequence library and pTarget plasmid backbone were ligated by Gibson assembly reaction, and then purified. Illustratively, ligation of each CiPGr library and pTarget plasmid backbone was achieved by 4 parallel Gibson assembly reactions. Thus, pTarget plasmid containing CiPGr sequence was obtained, and different individual host cells may contain different pTarget plasmids.
S20, ciPGr plasmids in the CiPGr plasmid library are transformed, transformed cells containing the CiPGr plasmids are screened, and the CiPGr plasmids are extracted from the transformed cells; and (3) 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.
In the step, ciPGr plasmids in the CiPGr plasmid library are transformed, so that the quantity of the CiPGr plasmids is enriched. In some embodiments, ciPGr plasmids from each CiPGr plasmid library are transformed into DH 5. Alpha. E.coli competent cells, transformed 2-10 times in parallel, and transformed DH 5. Alpha. E.coli competent cells are collected; spreading the DH5 alpha colibacillus competent cells on a culture dish containing chloramphenicol and kanamycin to culture, screening the DH5 alpha colibacillus transformed cells containing CiPGr plasmid, and extracting the CiPGr plasmid from the DH5 alpha colibacillus transformed cells.
Exemplary, the manner of plating DH 5. Alpha. E.coli competent cells on a culture dish containing chloramphenicol and kanamycin can be: incubated at 37 ℃ overnight.
And (3) 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. At this time, the host bacterium contains a two-plasmid system, i.e., pTarget plasmid and spCas9 plasmid. Wherein, the host bacteria containing the spCas9 plasmid is determined according to the type of the phage. Illustratively, when the phage contains at least one of tailed phages T7, T4, seszw and selz, the host bacterium containing the spCas9 plasmid can be escherichia coli (Mg 1655) containing the spCas9 plasmid and salmonella typhimurium containing the spCas9 plasmid.
The CiPGr plasmid-bacteria library obtained in the examples of the present application can be stored at-80 ℃.
In some embodiments, to assess coverage of a library of CiPGr sequences designed in a CiPGr plasmid-bacteria library, PCR against the CiPGr sequence library was performed and sequenced by HiSeq 2500 (Illumina). And (3) removing low-quality data from the obtained original sequencing data, comparing the sequencing data with the designed CiPGr library sequences, and determining the proportion of 100% consistency of each CiPGr library sequence in a plasmid library.
S30, culturing the wild phage and the corresponding CiPGr plasmid-bacterial library in a culture medium, transferring the mutant phage product to a fresh CiPGr plasmid-bacterial library for continuous culture, and repeating iterative culture to generate a mutant phage library.
In this step, the CiPGr plasmid-bacteria library is taken and added to the culture medium and cultured overnight. When the CiPGr plasmid-bacteria library is stored at-80 ℃, the CiPGr plasmid-bacteria library is thawed on ice and then added into a culture medium. Illustratively, the culture temperature is 37 ℃; illustratively, the medium may be LB medium. In some embodiments, to prevent contamination of the CiPGr plasmid-bacteria library by other bacteria, antibiotics and L-arabinose are added to the medium.
In one example, ciPGr plasmid-bacteria were added to LB medium and incubated overnight with antibiotics and L-arabinose.
Wild-type phage and the corresponding CiPGr plasmid-bacteria library are cultured in culture medium, phage infects the corresponding CiPGr plasmid-bacteria library, and phage DNA is injected into the cells of the corresponding CiPGr plasmid-bacteria library. And the gRNA encoded by pTarget in CiPGr plasmid-bacteria directs the spCas 9-encoded Cas9 nuclease to bind to the target gene on wild-type phage and double strand breaks the target gene. Repair by Homologous Recombination (HR) induced by homologous sequences results in the deletion or disruption of the phage gene. If the gene is not necessary for phage growth, progeny of the mutant phage can be amplified without the gene. Wild-type phage infects 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 this application, wild-type phage and the corresponding CiPGr plasmid-bacteria library were purified from mutant phage products produced after the first culture in culture medium to form a first generation mutant phage library.
It is understood that in the examples herein, the corresponding CiPGr plasmid-bacteria library refers to CiPGr plasmid-bacteria that wild-type phage are capable of infecting.
And (3) transferring the mutant phage product to a fresh CiPGr plasmid-bacterium library for continuous culture, so that the mutant phage product is transferred to fresh bacterium containing double plasmids, possibly infecting the bacterium containing different CiPGr plasmids, and deleting different genes. Through repeated iterative culture, the mutant phage product is continuously transferred to a new bacterium containing double plasmids, namely CiPGr plasmid-bacterium, so that gene deletion on the wild type phage genome is continuously accumulated. Meanwhile, the mutant strain with growth dominance can produce more offspring, and finally, the mutant strain takes a dominant position in a mutant population and becomes a dominant strain. In some embodiments, the iterative culture is repeated 300-600 times, thereby facilitating easy detection of non-essential, quasi-essential and essential genes.
It will be understood that reference to a fresh CiPGr plasmid-bacteria library in the examples of the present application refers to a CiPGr plasmid-bacteria library that has not been co-cultured with wild type phage and is not necessarily a freshly prepared CiPGr plasmid-bacteria library. It will be appreciated, however, that the type of CIPGr plasmid-bacteria library repeatedly grown iteratively, and the type of CIPGr plasmid-bacteria library during the first culture are identical for the same wild type phage.
In some embodiments, the generating step of the library of mutant phages is:
culturing wild phage and corresponding CiPGr plasmid-bacterial library in culture medium to generate first generation mutant phage product, centrifuging the first generation mutant phage product, and taking supernatant to obtain first generation mutant phage library; continuously culturing part of the first generation mutant phage library and a fresh CiPGr plasmid-bacterial 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 a fresh CiPGr plasmid-bacteria 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.
In this case, the mutant phage products are continuously transferred to new dual plasmid-containing bacteria, i.e., ciPGr plasmid-bacteria, and gene deletions are continuously accumulated on the wild-type phage genome, thereby forming mutant phages lacking different genes, and various mutant phages lacking different genes form a mutant phage library. Mutating various deleted genes in the phage library forms a set of deletable genes.
In the above method, each time a portion of the library of mutated phages obtained after cultivation was added to a fresh CiPGr plasmid-bacteria library, the other portion was stored at low temperature and further analyzed. In some embodiments, in the step of repeating the iterative culturing, the titer concentration of the mutant phage library is determined periodically; when the concentration of the obtained mutant phage is less than 10 5 At individual/ml, the mutant phage library was cultured using wild-type host cells. Since the number of mutant phages decreases due to passage, the number or the cell concentration of the mutant phages can be expanded by culturing the wild-type host cells in this case. Illustratively, the titer of the mutant phage library is determined every 10 transfers.
S40, sequencing a mutant phage library obtained after a certain transfer times, and confirming all genes which can be deleted in the phage.
In the step, a mutant phage library obtained after a certain number of transfer times is sequenced to obtain the deletable gene information of the mutant phage. In the step, all the stored mutant phages can be sequenced after the mutant phage library is obtained, and the obtained mutant phages can also be sequenced in the process of culturing the mutant phages.
In some embodiments, PCR is used to monitor the deletion of the phage gene once. Illustratively, one μ L of the mutant phage supernatant was used as a template for PCR, and Ex Taq DNA polymerase (Takara, RR01 AM) was used to amplify the target gene with appropriate primers, and the deletion of the gene was verified by agarose gel electrophoresis and Sanger sequencing.
In the examples of the present application, in order to monitor the efficiency of gene deletion, DNAs of mutant phage libraries were extracted according to the manual of phage DNA isolation kit (NORGEN, 46850), and DNA sequencing was performed using HiSeq1500 sequencer. Illustratively, the 20 th, 30 th, 40 th and 50 th adaptors of bacteriophages T7 and T4 are sequenced, and then sequenced every 50 to 100 adaptors.
In one possible embodiment, the methods provided in the examples herein also allow for the screening of a single active mutant phage. In some embodiments, screening a single 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 certain 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.
Illustratively, two libraries per phage (together forming 8 CiPGr plasmid-bacteria libraries) are exemplified by tailed phages T7, T4, seszw and selz. To obtain a single mutant phage, 1X 10 was used 3 The mutant phage library of PFU was mixed with 300. Mu.L of wild-type host cells and 10mL of 0.7% LB agar in one tube, poured onto a plate, and cultured overnight at 37 ℃. Randomly picking 8 large and 8 small plaques, and then separating and purifying. To determine the bactericidal effect of individual mutant phages, 10. Mu.l were usedAdding L of host cells cultured overnight and 200 mu L of fresh LB culture medium into a 96-well plate, purifying and picking up a single plaque into the 96-well plate, culturing on a microplate reader at 37 ℃, and detecting OD600 once every 10min for 12 hours; and selecting the mutant phage according to a sterilization curve, namely a phage one-step growth curve, and further purifying by a scribing method to obtain a single type of active mutant phage.
Exemplary, phage one-step growth curve assay methods are: after bacteria streaking, selecting monoclone for overnight culture; culturing phage, measuring concentration, and diluting to 10 5 PFU is reserved; preheating a culture medium, inoculating 10ml of the culture medium with 1% volume, culturing for 3 tubes in total at 37 ℃ for 2 hours, centrifuging at room temperature of 7000g, removing supernatant, adding 5ml of the preheated culture medium, and uniformly shaking; adding 0.2mmol/L CaCl, adding 100 mu L phage, shaking and mixing; standing at room temperature for 5min, adding preheated culture medium 30ml, shaking and mixing; after culturing for a certain time, sampling and continuously detecting the concentration.
Through the steps, the simplified phage genome with activity can be obtained rapidly and in high throughput, particularly for new phage with a large number of unknown functional genes.
In the process of carrying out iterative culture on the wild-type phage and the corresponding CiPGr plasmid-bacterium library, gene deletion on the wild-type phage genome is continuously accumulated, and meanwhile, the phage mutant with growth dominance can generate more filial generations and finally occupy the dominance in a mutant population to become a dominant strain.
In the embodiment of the application, the phage mutants are separated and subjected to monoclonal sequencing, so that a simplified phage genome can be obtained; and the mutant strains with stronger strength than wild phage can be separated by mixing the phage mutation libraries together for competitive culture. In one possible embodiment, the method provided in the examples herein can also be used to screen dominant mutant phages, the screening step comprising:
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-culturing, and repeating the step of transferring to fresh wild-type host cells for re-culturing for N-1 times to obtain TN mutant phages ((wherein T represents transfer, and N represents transfer number));
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 a single plaque into the 96-well plate, culturing on a microplate reader, and detecting OD once every other period 600 Lasting for 12 to 24 hours; and (4) screening dominant mutant phages according to the bactericidal curve.
Illustratively, for the tailed phages T7, T4, seszw and selz, the different adapted mutant phage pools were mixed together, a mixed mutant phage pool (10) 5 PFU) and corresponding logarithmic growth (10) 8 PFU) for 2 hours to obtain T1 mutant phage; and transferring part of the T1 mutant phage to a fresh host cell for culture, so that 8 times of transfer are carried out in total, and 16-20 generations of amplification are carried out to obtain the T8 mutant phage. Will 10 3 PFU of T8 mutant phage, 300. Mu.L of wild type host cells and 10mL of 0.7% LB agar were added to one tube, mixed, poured into LB plate, and cultured overnight at 37 ℃.3 different plaques were picked for purification. To determine the bactericidal effect of mutant phages alone, 10 μ L of overnight cultured host cells, 200 μ L of fresh LB medium were added to 96-well plates, and individual plaques were purified and transferred to 96-well plates, cultured on a microplate reader at 37 ℃, and detected at OD600 every 10min for 12 hours.
According to the embodiment of the application, the nonessential genes, the quasi-essential genes and the essential genes can be simply and conveniently detected by performing metagenome sequencing on a mutant phage library. In one possible embodiment, the methods provided in the examples herein further comprise the determination of non-essential, quasi-essential, and essential genes in the bacteriophage.
In some embodiments, the determination of non-essential, quasi-essential, and essential genes in a bacteriophage comprises:
analyzing the probability of the deletion gene of the mutant bacteriophage in the mutant bacteriophage library, wherein the deletable gene with the deletion frequency of less than 5 percent is determined as the quasi-essential gene of the bacteriophage, the deletable gene with the deletion frequency of more than 5 percent is determined as the non-essential gene of the bacteriophage, and the gene without the detected deletion of the deletable gene is determined as the essential gene of the bacteriophage.
It is understood that the frequency of a gene mutation is expressed as a percentage of gene deletions/gene retentions in a mutation library. In the process of carrying out iterative culture on wild-type phages and the corresponding CiPGr plasmid-bacterial libraries, gene deletions on the wild-type phage genome are continuously accumulated, and phage genes can be preliminarily classified into three groups according to the results of iterative culture: (1) Genes deleted in the isolated single mutant phage genome with a relatively high frequency of gene deletion, more than 5%, were classified as non-essential genes. (2) Other genes in the set of deletable genes with a frequency of gene deletions <5% are classified as quasi-essential genes. Deletion of these genes results in defective phages that grow less favorably than mutants that delete nonessential genes. (3) Genes not detected in the set of deletable genes are important for phage growth and are classified as essential genes.
Exemplarily, in the case of tailed phages T7, T4, seszw and selz, the majority of the detected gene deletions (T7, T4.4%, seszw94.1%, selz 98.4%) in the isolated individual mutant phage genomes showed a relatively high frequency > 5% in the transferred mutant phage libraries, indicating that these genes are less important for phage growth than other genes (< 5% in frequency).
The embodiment of the application can obtain the minimal phage genome and the phage with stronger bactericidal capacity by identifying the essential genes, the nonessential genes and the quasi-essential genes of the phage.
It will be appreciated that eukaryotic viruses may also be used to simplify the genome by high throughput deletion of genes by reference to the methods provided in the examples herein. In the method, the phage is correspondingly replaced by the eukaryotic virus, the host bacteria is correspondingly replaced by the host cell, and the CiPGr plasmid-bacteria is correspondingly replaced by the CiPGr plasmid-cell.
Referring now to FIG. 2, the tailed phages T7, T4, seszw and selz are used as examples to illustrate the phage genome simplification process, the selection and determination of non-essential genes and quasi-essential genes in the phage genes, and the formation and selection of dominant strains, in combination with specific examples.
(1) CiPGr plasmid library design, construction and transformation
A total of 8 CiPGr sequence libraries of 200bp, 2 CiPGr sequences per phage, were designed for the potential deletable gene sequences of tailed phages T7, T4, seszw and selz. Wherein, the sequence of CiPGr is shown in figure 2-a, and comprises a bar code (20 bp), a homology arm (50 multiplied by 2 bp), a promoter (36 bp), a spacer (20 bp) and a primer (24 bp).
As shown in FIG. 2-c-1, all designed CiPGr libraries were synthesized on a 6000-size DNA chip. Cassette-100 (disrupted gene) and cassette-gene cassettes (deleted gene) of 4 kinds of phages were isolated by PCR under the following cycle conditions: 2 minutes at 98 ℃;98 ℃,10s,58 ℃,20s,72 ℃,6s; the mixture was stored at 72 ℃ for 10 minutes and 4 ℃.
As shown in FIG. 2-c-2, pTarget plasmid backbone was amplified by PCR and the plasmid backbone and CiPGr library were purified by gel electrophoresis. Each CiPGr sequence library was assembled with pTarget plasmid backbone by 4 parallel Gibson assembly to form a CiPGr plasmid library, which was then purified. Transforming the purified CiPGr plasmid library into DH5 alpha escherichia coli competent cells, parallelly transforming each library for 2-10 times, and collecting the transformed DH5 alpha escherichia coli competent cells; competent cells of DH 5. Alpha. E.coli were plated at 15cm 2 To the petri dish, chloramphenicol and kanamycin were added, and the culture was performed overnight at 37 ℃. After the colonies were scraped from the culture dish, ciPGr plasmid was extracted from the transformed cells of DH 5. Alpha. E.coli. Transforming the 8 CiPGr plasmid libraries into escherichia coli (Mg 1655 containing spCas9 plasmid) and salmonella typhimurium (containing spCas9 plasmid) to obtain CiPGr plasmid-bacterium libraries, and storing the CiPGr plasmid-bacterium libraries inThe mixture is kept at-80 ℃ for later use.
In this step, to assess the coverage of the CiPGr libraries designed in the plasmid library, PCR against the CiPGr library was performed and sequenced by HiSeq 2500 (Illumina). And removing low-quality data from the obtained original sequencing data, comparing the sequencing data with the designed CiPGr library sequences, and determining the proportion of 100% consistency of each CiPGr library sequence in a plasmid library.
(2) Generation of mutant phage libraries and metagenomic sequencing
The libraries containing CiPGr plasmid-bacteria stored at-80 ℃ were removed, and after thawing on ice, 300. Mu.L of each library was removed and added to 15ml of LB medium together with antibiotics and L-arabinose, and cultured overnight at 37 ℃. FIG. 2-c-3,1ml phage (10) 9 PFU/ml), 1ml of corresponding host cells containing a plasmid library (10) 9 CFU/ml) and 1ml LB medium were added to a shake tube and cultured at 37 ℃ for 6 hours to produce the first generation mutant phage product. Taking 1mL of the first generation mutant phage product, centrifuging, taking supernatant, and transferring to 1mL of fresh CiPGr plasmid-containing bacterial library and 1mL of LB culture medium for culture; the transfer was performed 300-600 times as described above. FIG. 3 provides a library of phage deletable genes showing the frequency of various gene deletions in the nth-time-transferred phage mutation library. The deletable gene library was generated by the transposition of two types of plasmid libraries (gene disruption library, gene deletion library), where the frequency represents the percentage (log 2) of the corresponding reads (deletion or disruption) of the gene in the population and the corresponding heatmap was generated by the phantom R software package. "L" indicates the deletion of a large fragment that we did not design; "P" means the deletion of one or two bases of the coding region, resulting in premature termination of the codon.
Among these, 1ml of the supernatant produced in each transfer was used for passaging, and 1ml was stored at 4 ℃ for further analysis. The titer of the mutant phage pool was determined every 10 transfers. If the concentration of the mutant phage is less than 10 5 Per ml, wild-type host cells were used to culture the mutant phage library.
The phage gene deletion was monitored by PCR every 10 transfers. mu.L of the supernatant was used as a template for PCR, and amplification was performed on the target gene using Ex Taq DNA polymerase (Takara, RR01 AM) and appropriate primers. Gene deletion was verified by agarose gel electrophoresis and Sanger sequencing. As shown in FIG. 4, PCR is provided to monitor the deletion of the phage gene, 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.
To monitor the efficiency of gene deletion, the DNA of the mutant phage library was extracted and sequenced using a HiSeq1500 sequencer according to the manual of the phage DNA isolation kit (NORGEN, 46850). We sequenced the 20 th, 30 th, 40 th and 50 th adaptors of phages T7 and T4, and then every 50 to 100 adaptors.
(3) Screening, characterization and genomic sequencing of individually isolated mutant phages
As shown in FIG. 2-c-4, to obtain individual mutant phages, 10 3 The mutant phage library of PFU was mixed with 300. Mu.L of wild-type host cells and 10mL of 0.7% LB agar in one tube, poured into a plate, and cultured overnight at 37 ℃. Randomly pick 8 large and 8 small plaques, which are then isolated and purified.
To determine the bactericidal effect of individual mutant phages, 10 μ L of overnight cultured host cells, 200 μ L of fresh LB medium were added to 96-well plates and individual plaques were purified and picked into 96-well plates. Culturing on a microplate reader at 37 deg.C, and detecting OD600 every 10min for 12 hr; mutant phages were selected according to the bactericidal curve, i.e. the phage one-step growth curve, and further purified by the scratching method. The method for measuring the phage one-step growth curve comprises the following steps: after bacteria streaking, selecting single clone for overnight culture; culturing phage, measuring concentration, and diluting to 10 5 PFU is reserved; preheating a culture medium, inoculating 10ml of the culture medium with 1% volume, culturing for 3 tubes in total at 37 ℃ for 2 hours, centrifuging at room temperature of 7000g, removing supernatant, adding 5ml of the preheated culture medium, and uniformly shaking; adding 0.2mmol/L CaCl, adding 100 mu L phage, shaking and mixing; standing at room temperature for 5min, adding preheated culture mediumCulturing 30ml of culture medium, shaking and mixing uniformly; after culturing for a certain time, sampling and continuously detecting the concentration.
FIG. 5 shows plaques of mutant phage T7 in double-layered agar plates; FIG. 6 is a bactericidal curve for mutant phages T7 and T4 against MG1655 and for mutant phages seszw and selz against Salmonella ST 56.
(4) Screening, characterization and genome sequencing of more strongly mutated phages
Mixing together different adapted mutant phage libraries, mixed mutant phage libraries (10) 5 PFU) and corresponding logarithmic growth (10) 8 PFU) for 2 hours to obtain T1 mutant phage; and transferring part of the T1 mutant phage to a fresh host cell for culture, so that 8 times of transfer are carried out in total, and 16-20 generations of amplification are carried out to obtain the T8 mutant phage. Will 10 3 PFU of T8 mutant phage, 300. Mu.L of wild type host cells and 10mL of 0.7% LB agar were added to one tube, mixed, poured into LB plate, and cultured overnight at 37 ℃.3 different plaques were picked for purification. To determine the bactericidal effect of mutant phages alone, 10 μ L of overnight cultured host cells, 200 μ L of fresh LB medium were added to 96-well plates, and individual plaques were purified and transferred to 96-well plates, cultured on a microplate reader at 37 ℃, and detected at OD600 every 10min for 12 hours.
FIG. 7 is the bactericidal curve for the selected stronger phages, where a is the bactericidal curve for stronger T7 and T4 mutants against MG1655 and stronger seszw and selz mutants against Salmonella (ST 56); b is the one-step growth curve of stronger phages for T7, seszw, T4 and selz. In the figure, WT represents a wild-type phage; data are presented as mean ± SD of three experiments; phage titers were determined by the PFU method.
FIG. 8 is a genetic map of the selected stronger phage in which black bars indicate the region of gene deletion; black dots indicate point mutations. The arrows indicate genes, and the direction of the arrows corresponds to the direction of transcription and translation.
(5) Data analysis
The frequency of a gene mutation is expressed as the percentage of gene deletions/gene retentions in a mutation library. We used the open source software breseq (version 0.28.0) to predict point mutations and gene deletions for each mutation library; the SOAP-denove (V2.04-r 241) software was used for genome assembly of the mutant phages.
The application of CiPGr to four different tailed phages (model phages T7 and T4; salmonella phage seszw and selz) resulted in the deletion of 8-23% (3.3-35 kbp) of sequences from mutants of these phages, resulting in a simplified phage backbone. Macro-set sequencing of the mutant phage library showed that the nonessential and quasi-essential genes accounted for 46.7% -65.4% of the total number of these phage genes. The loss of the quasi-essential gene (24% to 26%) may cause severe damage to the phage amplification, causing the corresponding mutant to fade out in the mutant library, resulting in the deletion of the quasi-essential gene not being detectable in the genome of the isolated single mutant.
Mutant phages with stronger bactericidal capacity than wild-type phages were obtained by screening. We observed that the selected stronger mutants performed faster than their wild-type phage in killing the host (T7 5min faster, T4 2h faster, seszw 1 h faster, selz 2h faster).
The above results indicate that CiPGr is a versatile and efficient method, applicable to novel phages and other eukaryotic viruses without any prior knowledge.
The above are merely alternative embodiments of the present application and are not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement or the like made within the spirit and principle of the present application shall be included in 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.
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