CN114134091A

CN114134091A - Identification method and application of bacterial genome insulation site

Info

Publication number: CN114134091A
Application number: CN202111317839.4A
Authority: CN
Inventors: 胡杨波; 何丽娜; 陈士云
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2022-03-04
Anticipated expiration: 2041-11-08
Also published as: CN114134091B

Abstract

The invention discloses an identification method and application of a bacterial genome insulation site. The invention realizes that the exogenous gene is inserted into the insulating expression site of the bacteria, so that the expression of the exogenous gene does not influence the expression of other genes of the bacteria. Provides a new idea for the modification and application of probiotic escherichia coli EcN and other bacteria for integrating gene lines.

Description

Identification method and application of bacterial genome insulation site

Technical Field

The invention relates to the field of microbial synthetic biology, in particular to an identification method and application of a bacterial genome insulation site.

Background

Synthetic biology is used as a new interdiscipline, integrates technologies in the fields of genetic engineering, biochemistry, system biology, computer biology and the like, and is used for editing and reconstructing live vectors so as to endow the live vectors with specific physiological functions. With the enrichment of genomics, proteomics, metabonomics and phenomics theories and the development of related technologies, the regulation and reconstruction of host gene expression, and the precise design of target gene expression lines to create different functional living cells have become the main targets of synthetic biology. Among them, microbial cells become the first choice of living factories due to their simple genetic background and mature editing technology, and provide a good base for the field of biotherapy of living organisms.

Therefore, based on the development of microbial synthetic biology, people try to find new therapeutic macromolecules, and at the same time, a series of editing technologies are used for directionally modifying some engineering strains to realize the in-vivo delivery of the macromolecules, particularly for the editing modification of intestinal probiotics, namely escherichia coli Nissle 1917(EcN) and Lactobacillus (LAB), the safety of the macromolecules is proved, but the self-therapeutic effect is limited, and the function and application value of the macromolecules need to be widened by means of synthetic biology. The research has proved that the lactobacillus LAB introduces IL-10 with secretion expression to effectively endow the lactobacillus LAB with the function of treating inflammatory enteritis (IBD), EcN genome is edited, multiple copies of the gene express and deliver phenylalanine amino lyase to treat phenylketonuria, the EcN arginine metabolic pathway is regulated to obviously improve the survival rate of the mouse with high blood ammonia, and the strains enter the clinical test period at present. Besides, EcN delivery matrix fiber is used for treating intestinal mucosa injury, a quorum sensing system is used for controlling EcN gene circuit for expressing and inhibiting pathogenic bacteria, mucosal immunity is stimulated by means of presenting specific antigens of escherichia coli, and the like, most of the engineering strains are based on plasmid expression foreign protein, but two important risks exist in a mode of carrying active molecules through plasmids, namely instability of functions of the plasmids in vivo and threat of resistance genes to macroscopic environment, and therefore the exogenous fragment expressed by using genome is more in line with editing criteria of bioactive vectors in the aspects of maintaining genetic stability and biological safety.

Insertion of foreign fragments into the genome can reduce the metabolic burden of the bacterium, but can cause impaired protein expression and to a large extent affect the transcriptional levels of other genes in the bacterium, even alter the properties of the bacterium itself. To this end, there are studies to identify the presence of three insulating "landing pads" (mapping pads), pstS/glmS, thic/rsd, ythA/fecE on the e.coli MG1655 genome by way of transposable library, which provide better integration sites for the engineering of the commonly used engineered strain e.coli MG1655, but whether the insulating sites searched by this method are suitable for other strains has never been reported. At present, for the modification of EcN, malEK, malPT, yicS/nepI and the like are often selected as exogenous gene insertion sites, most of the sites are located in an operon, partial evidence shows that the insertion of genes into the sites has a great influence on the level of the whole bacterial transcriptome, and the safety and the applicability of the sites are still to be researched. Therefore, a set of novel methods for searching for insulating sites on bacterial genomes is discovered and established, and the method has important significance for the subsequent modification and application of microorganisms in the field of synthetic biology.

Disclosure of Invention

The invention aims to explore and identify insulating sites which can be used for inserting exogenous genes on the genome of a common engineering strain in the field of synthetic biology so as to expand the application value of the engineering strain in the synthetic biology. Based on the conservation of bacterial genes and the common characteristics of bacterial transcription termination, the present invention may, in some embodiments, select escherichia coli e.coli MG1655, e.coli DH5 a; examples of the strain include industrially commonly used strains such as e.coli DH10 β and e.coli bl21(DE3), and probiotic strains such as lactococcus lactis (lactococcus lactis), bifidobacterium (bifidobacterium bifidum), bacteroides fragilis (bacteroides fragilis), Bacillus coagulans (Bacillus coagulosus), and Streptococcus thermophilus (Streptococcus thermophilus) may be selected.

To achieve the above object, the present invention provides a method for identifying an insulating site in a bacterial genome, which is a region in the genome where the transcription level is low and does not affect or less affects the expression of surrounding genes, comprising the steps of: and (3) determining that the two ends of the site to be detected have strong transcription terminators by a biological method, and the ends of the two strong transcription terminators are opposite, namely identifying the site as an insulating site.

In the above embodiment, the bacterium may be any bacterium such as Escherichia coli, but is not limited to the above-described exemplary species, and probiotic Escherichia coli (Escherichia coli) Nissle 1917 is preferable.

Preferably, the insulating site is a sequence located in the middle of the uspG and aphF genes in E.coli.

In the above scheme, the sequence corresponding to the insulated site is transcribed into mRNA secondary structure according to the characteristics of the transcription terminator. Bioinformatics methods analyze mRNA transcription terminator secondary structures as hairpin structures and strong transcription terminators conform to the characteristics of the hairpin structure followed by more than 5 contiguous U nucleotides.

For example, the above identification method can be realized by the following steps:

(1) firstly, visual statistics is carried out by using the existing transcriptome data of the targeted bacterial strain, and a non-transcription region with expression hardly changing along with conditions is searched. In the present invention, we take probiotic EcN as an example, find a region without transcription in its genome between the genes uspG (SEQ. ID. NO:003) and ahpF (SEQ. ID. NO:004) (FIG. 1), and provide the sequence information of the region on the genome SEQ. ID. NO: 001;

(2) after determining the non-transcription region on the genome, further searching sequence regions with insulating expression site characteristics by RNA secondary structure prediction software, namely, the two ends of the site have transcription strong terminator structures facing to the insulating site. For example, the present invention illustrates EcN in which a Highly-insulated Site (HIS-1) has distinct transcription terminator structures at both ends (FIG. 2) and provides its specific positional information and sequence information (SEQ. ID. NO: 005);

(3) after the insulated site meeting the conditions is screened, a gene editing means is used for editing and transforming the site, a foreign gene segment comprising a promoter, a coding region and a transcription terminator region is inserted, and then the influence of the insulated site expression foreign gene on the physiological function and the gene expression of the bacteria is verified through phenotype verification and transcriptomics verification. In the specific embodiment of the invention, by taking the HIS-1 of EcN as an example for expressing the uricase gene from candida, growth phenotype, transcriptome analysis and the like are utilized to prove that the strategy can realize the insulating expression of the exogenous gene;

(4) can perform functional high expression on the basis of exogenous insertion gene insulation expression, and provides a reasonable modification strategy for expanding bacteria into a chassis in the field of synthetic biology. In the specific embodiment of the invention, a modified bacterium EcN UA for expressing a candida-derived uric acid oxidase gene at a probiotic EcNHIS-1 site is provided, and the modified bacterium EcN UA is proved to have a function of degrading uric acid in vitro.

The invention also provides a method for preparing the recombinant bacteria, which inserts the exogenous gene into the insulating site of the bacteria, so that the expression of the exogenous gene does not influence the expression of other genes of the bacteria; the insulated site is a site which is provided with strong transcription terminators at two ends and the tail ends of the two strong transcription terminators are opposite.

In the above protocol, the insulating site may be located on the bacterial chromosome or on a plasmid. The inserted exogenous gene can be any gene, but the gene is required to be provided with basic elements such as a promoter, a coding gene, a transcription terminator and the like, so that the normal expression of the exogenous gene is ensured. Preferably expresses metabolic related genes such as urate oxidase and the like, and meets the sequence characteristics of SEQ ID No. 002. The verification that the expression of the exogenous gene does not influence the expression of other genes of the bacteria is mainly reflected in that the analysis of phenotype, transcriptomics and the like of the bacteria after the exogenous gene is introduced does not show overall obvious difference.

Also in the above embodiment, the bacteria may be selected from Escherichia coli, lactococcus lactis, Bifidobacterium, Bacteroides fragilis, Bacillus coagulans or Streptococcus thermophilus, but not limited thereto. Preferably, the bacterium is escherichia coli. More preferably, the insulating site is a sequence located in the middle of the uspG and aphF genes in E.coli.

Optionally, the exogenous gene is introduced in a manner selected from the group consisting of homologous recombination exchange, CRISPR gene editing, and phage transduction. Techniques such as homologous recombination and CRISPR gene editing that do not introduce a resistance gene are preferable.

The invention also provides a recombinant bacterium, which comprises the insulation site and an exogenous gene inserted into the insulation site.

The invention also discloses application of the identification method of the bacterial genome insulation site in a bacterial gene editing technology.

The invention has the beneficial effects that:

by the discovery strategy and application of the bacterial genome insulation site, the insertion of the exogenous gene into the bacterial insulation expression site is realized, so that the expression of the exogenous gene does not influence the expression of other genes of bacteria. Provides a new idea for the modification and application of probiotic escherichia coli EcN and other bacteria for integrating gene lines.

Drawings

FIG. 1 shows the transcription and position information of the region around the HIS-1 site under different conditions.

FIG. 2 shows the results of the secondary structure prediction of the HIS-1 double-sided terminator.

FIG. 3 shows the result of exogenous expression of urate oxidase at HIS-1 site.

FIG. 4 is a growth curve of the engineered strain EcN UA.

FIG. 5 shows the bactericidal phenotype results of the engineered EcN UA strain.

FIG. 6 shows the transcriptome comparison of the engineered EcN UA strain with the non-engineered strain.

FIG. 7 is an analysis of the effect of foreign gene insertion in the engineered strain EcN UA on the transcription of genes surrounding HIS-1.

Detailed Description

The invention is described in further detail below with reference to the figures and specific embodiments. The following examples are carried out on the premise of the technical scheme of the invention, and detailed embodiments and specific operation procedures are given, but the scope of the invention is not limited to the following examples.

The invention preferably provides a method for searching transcription insulation sites on a bacterial genome, the prokaryotic bacterial genome is continuous, and the research on transcription background is clear, so that the method for searching the transcription insulation sites on the genome by using bacterial related transcriptome data has universality. In the embodiment, probiotic escherichia coli EcN is preferably selected as a research object, EcN related transcriptome data is analyzed by means of bioinformatics, a landing pad with a lower transcription level is searched, the expression conditions of surrounding genes are counted, RNA secondary structure prediction software is used for discovering that a natural double-terminator structure exists in the landing pad, and a set of method for searching genome insulation sites is established. Then introducing exogenous genes into the site, verifying that the target gene realizes functional expression through transcriptome data and in vitro enzyme activity experiments, ensuring that the site has high insulativity and has very little influence on the whole transcription of bacteria, simultaneously proving that the basic characteristics of the modified strain are not changed and realizing specific functions through the characterization analysis of the modified strain, and providing reference for the application of the genetic modification EcN in the field of synthetic biology.

The following examples were performed with the primary focus on transcriptome data associated with probiotic E.coli EcN. In some embodiments, the conditions under which the transcriptome library is constructed may be a change in environmental stress, or may be a change involving overexpression or knock-out of a particular gene.

Screening for regions with no significant transcription under various conditions by analyzing EcN transcriptome changes under various conditions and defining the regions as "transcriptional insulator" (SEQ. ID. NO: 001);

after the transcribed insulating region is finalized, the sequence is predicted by using RNA secondary structure online prediction software (mFold), two natural terminators T1 and T2 which are positioned at the 3' end of the uspG and ahpF genes are determined to exist in the region, the sequence information is respectively SEQ ID No. 003 and SEQ ID No. 004, and the structure is shown in FIG. 2. Definition the 8bp sequence between T1 and T2 is defined as a Highly insulating Site (HIS-1) with sequence information of seq.id No. 005;

inserting exogenous gene uricase with sequence information of SEQ.ID.NO. 002 into HIS-1 site to obtain recombinant strain EcN UA for verifying the insulativity of the site. In some embodiments, the exogenous gene may be integrated into the gut probiotic genome by genetic engineering techniques (including homologous recombination, gene editing, etc.).

Examples the phenotypic assay of the engineered strains was by growth curve reaction. In some embodiments, the bacterial phenotype is identified by a bacterial colony morphology reaction, and may also be identified by a microscopic reaction to a change in the bacterial microstructure morphology.

The influence of the site on the global gene of the bacterium after the exogenous gene is expressed is analyzed by comparing the EcN UA and EcN transcriptome data, and the result shows that the expression of most of the genes of the modified strain is consistent with the EcN wild type and is in a good linear relation, which indicates that the insertion of the site gene does not influence the gene of the bacterium, and the insulation property is high.

The in vitro enzymatic kinetics curve verifies that the modified strain EcN UA can highly express the exogenous gene uricase and has better function. In some embodiments, the expression of the gene of interest can be detected from mRNA levels by fluorescent quantitative PCR reactions, and protein expression levels can also be detected by Western Blot.

The experimental process provides an insulating site HIS-1 which can be used for exogenous genetic circuit integration on the genome of probiotic escherichia coli EcN, and obtains a modified recombinant strain EcN UA to confirm the insulativity and practicability of the site, thereby providing reference for genetic modification and application of EcN.

Example 1

Analysis of transcriptome-free regions on genomes Using bacterially-related transcriptome data

The genome of prokaryotic bacteria is continuous, and the regulation and control function network among genes is complex and variable, so that the interference of foreign genes on host bacteria is avoided when target gene fragments are introduced into the chassis genome by a synthetic biology means. The transcriptome data of the bacteria can intuitively reflect the transcription activity level of different positions of the bacterial genome, so that the transcriptome data can be used as a basis for searching a genome transcription insulated site. In this example, we used probiotic E.coli EcN as a subject, adjusted EcN-related transcriptome data ERR:2983261, SRR:11357513, SRR:6658275, SRR:9994133 in NCBI Sequence Read Archive (SRA) database, and analyzed the transcription levels of different genes in these transcriptomes under respective conditions using R (v3.2.2) visually, and found a no-transcription region after the stop codons of two genes in opposite directions, usp G (SEQ. ID. NO:003) and ahpF (SEQ. ID. NO:004) (FIG. 1), and initially analyzed the insertion site (SEQ. ID. NO:001) that could be the isolated expression of the foreign gene on EcN genome.

Relevant software used in the examples:

NCBI SRA database：https://www.ncbi.nlm.nih.gov/sra

transcriptome data analysis Using R (v3.2.2)

Example 2

Discovery of double terminator structure around untranscribed region and identification of insulated expression site

In example 1 we found that some special elements may be present in the "transcriptional insulator" (SEQ. ID. NO:001) that lead to its transcriptional insulation, so in this example we first analyzed this sequence using RNA secondary structure on-line prediction software (mFold) and showed that there are two genomic natural terminator structures T1(SEQ. ID. NO:003) and T2(SEQ. ID. NO:004), T1 responsible for the transcriptional termination of uspG, T2 responsible for the transcriptional termination of ahpF (FIG. 2), such a double terminator structure can provide a natural "transcriptional insulator" as an integration Site for genetic engineering and this natural structure has a universal (Precision design of stable genetic engineering in high-engineered E.coli genetic engineering) in the bacterial genome, this sequence has a high sequence spacing between T9584 (T1. and T598) that we defined this high sequence as SEQ ID-005 bp-insert Bio56, HIS-1)

Relevant software used in the examples:

mFold：http://www.unafold.org/

example 3

Construction of HIS-1 site integration exogenous gene uricase of intestinal probiotic EcN genome

In this example, a UA coding gene (SEQ. ID. NO:002) carrying Candida origin combined with a transferred suicide plasmid pDM4 was constructed, an artificially optimized strong promoter P6 (SEQ. ID. NO:006) was introduced into the upstream of the gene, a strong terminator rrnBT (SEQ. ID. NO:007) was added into the downstream of the gene, and the above elements were knocked into and integrated with the HIS-1 site on the genome of Escherichia coli EcN by homologous recombination (FIG. 4), thereby obtaining a recombinant strain EcN UA.

The specific implementation steps are as follows:

1. EcN strain carrying a temperature sensitive plasmid pKD46(Amp) was constructed. The pKD46 plasmid (One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products. ProcNatlAcadSci U S A.2000,97, 6640-containing 6645) was electroporated EcN by an electrotransfer instrument (EppdrfffElectroporator 2510) and cultured at 30 ℃ to give EcN strain carrying Amp resistance.

Construction of pDM 4-UAm. A recombinant plasmid pDM4-UAm plasmid is constructed by taking a pDM4 suicide plasmid (Flagellin A is an infectious plasmid for the virus of Vibrio anguillarum.J.bacteriol.1996, 178,1310-1319) as a starting vector, and carries 750bp each of a uricase and an upstream and downstream homology arm of HIS-1.

EcN UA strain construction. The pDM4-UAm plasmid is introduced into EcN bacteria transformed with pKD46 plasmid by means of conjugative transfer, spread on Amp + Cm double-resistant LB plate, and cultured overnight at 30 ℃ to obtain single-exchange strain. The single-crossover clone was streaked onto a salt-free LB + 10% sucrose plate and cultured at 40 ℃ to obtain a double-crossover mutant from which the pKD46 plasmid was removed, which was designated as EcN UA.

Media and reagents used in the examples:

LB culture medium:

adding into 1L of distilled water

Peptone: 10g (OXOID cat # LP 0042);

10g of NaCl (national drug cargo number 7647-14-5);

yeast powder: 5g (OXOID cat # LP0021),

autoclaving at 121 ℃ for 20 minutes.

Sucrose (national drug delivery number 57-50-1)

Ampicillin Amp (Shanghai Yuan leaf biotechnology Co., Ltd.; product No. 69-52-3)

Chloramphenicol Cm (BIOBASICINC cat No. 56-75-7)

Example 4

Modified strain EcN UA growth isophenotypic test result

In example 3, we used homologous recombination techniques to seamlessly insert the foreign gene, uricoxidase, at the HIS-1 site. In this example, the phenotypic change of the strain EcN UA was modified by measuring the growth curve response.

EcN and EcN UA monoclonals were picked in triplicate and incubated overnight in LB non-resistant liquid. 1: 100 were inoculated into fresh LB medium at 37 ℃ and sampled at specific time points to determine OD 600. The results show that the engineered strain EcN UA grew without difference compared to the non-engineered strain (fig. 4), confirming that the modification of the HIS-1 site did not affect the bacterial growth phenotype.

In addition to the validation of the growth phenotype, we also performed phenotypic validation for the EcN-specific phenotype, the target salmonella typhimurium LT 2. Firstly EcN and EcN UA carrying Kan resistant plasmid are transformed, LT2 is transformed with Cm resistant plasmid, and transformant is selected to be cultured in LB corresponding resistant liquid overnight. 1: 100 were inoculated into fresh LB resistant liquid supplemented with 0.2mM DIP to simulate a low iron environment and incubated overnight at 37 ℃. Then switching to about 10³The CFU bacteria are added into 100 mul of fresh DMEM/F12 low-iron culture medium, independent culture and co-culture combination are carried out, another part is supplemented with 1 mul of Fe (III) and used as a control, after 8 hours of co-culture, 10 times of gradient dilution is carried out, and counting and spot spotting are carried out on respective resistance screening plates. The results show that under the condition of low iron, EcN UA is consistent with EcN wild type phenotype, and both show good bactericidal activity (FIG. 5), which indicates that the integration of exogenous genes at HIS-1 locus has no influence on EcN bactericidal properties.

Media and reagents used in the examples:

LB medium as in example 3;

chloramphenicol Cm (BIOBASICINC cat No. 56-75-7)

Kanamycin Kan (Anniji chemical, cat number 25389-94-0)

DIP (sigma, cat number 112246-73-8)

DMEM/F12(Thermo Fisher, cat # 11320033)

Enzyme mark instrument (BioTek)

Example 5

Modified probiotic EcN UA RNA-Seq analysis

In order to analyze the influence of the expression of foreign genes introduced into the HIS-1 site on the bacterial transcription level, a transcriptome library of EcN and EcN UA is constructed for analyzing the insulation of HIS-1. The specific implementation process is as follows:

1. EcN and EcN UA monoclonal colonies were picked in triplicate and cultured overnight in non-resistant LB liquid, following a 1: 100 is inoculated into 4ml of fresh LB for culturing for 2.5h, the temperature is 37 ℃, and the rpm is 200;

2.5000rpm, 5min to collect 4ml of bacteria, remove supernatant, extract sample RNA using TRIzol according to the instructions, remove rRNA using Ribo-off rRNA deletion Kit, use

The UltraTM directed RNA Library Prep Kit was used for Library construction with reference to the instructions, sample sequencing using the IlluminaHiSeq X platform and transcriptome data analysis using R (v 3.2.2).

The analysis result of the transcriptome shows that compared with the unmodified strain, uricase inserted into the HIS-1 locus has higher expression level, and meanwhile, the comparison of the EcN UA and EcN overall transcription levels shows that the transcription of EcN overall genes is not influenced except that the expression levels of a few genes in the EcN UA transcriptome are obviously changed (figure 6), and the insertion of foreign genes does not influence the transcription levels of uspG and ahpF of upstream and downstream genes of the HIS-1 locus (figure 7), so that the HIS-1 is proved to have high insulating property and to be suitable for the insulating integration of the foreign genes.

Media and reagents used in the examples:

LB medium as in example 3;

TRIzol (Invitrogen, cat # 15596018)

Ribo-off rRNA deletion Kit (Vazyme, cat # N407-01)

UltraTM directed RNA Library Prep Kit (NEB, cat # E7420L).

Sequence listing

<110> Wuhan Virus institute of Chinese academy of sciences

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Met Glu Ala Thr Val Thr Cys Leu Leu Glu Gly Gly Phe Asp Thr Ser

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Met Phe Tyr Gly Tyr Asn Lys Cys Asp Phe Thr Thr Leu Gln Pro Thr

165 170 175

Thr Asp Arg Ile Leu Ser Thr Asp Val Asp Ala Thr Trp Val Trp Asp

180 185 190

Asn Lys Lys Ile Gly Ser Val Tyr Asp Ile Ala Lys Ala Ala Asp Lys

195 200 205

Gly Ile Phe Asp Asn Val Tyr Asn Gln Ala Arg Glu Ile Thr Leu Thr

210 215 220

Thr Phe Ala Leu Glu Asn Ser Pro Ser Val Gln Ala Thr Met Phe Asn

225 230 235 240

Met Ala Thr Gln Ile Leu Glu Lys Ala Cys Ser Val Tyr Ser Val Ser

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Tyr Ala Leu Pro Asn Lys His Tyr Phe Leu Ile Asp Leu Lys Trp Lys

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ccggggtcag ccagcctgag cctgcaccgt tttgccgctg atgtacgtcg ttttgaagag 180

catctgcaac atgaagcaga agaacgtctg caaacgatgg tcagccactt caccatcgat 240

ccttcccgca ttaagcaaca tgtccgtttt ggtagtgtgc gggatgaagt taacgagtta 300

gcgaaagaac ttgatgcaga tgtagtagtg attggttcgc gtaacccatc aatttcaact 360

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gctgaaatcg cagaactgtc agacaaagtc acctttaaag aagataacag cttgccggtg 180

cgtaagccgt ctttcctgat caccaaccca ggttccaacc agggaccacg ttttgcaggt 240

tccccgctgg gccacgagtt cacctctctg gtactggcgt tgttgtggac cggtggtcat 300

ccgtcgaaag aagcgcagtc tctgctggag cagattcgcc atattgacgg tgattttgaa 360

ttcgaaacct attactcgct ctcttgccac aactgcccgg atgtggtgca ggcgctgaac 420

ctgatgagcg tactgaaccc gcgcatcaag cacactgcaa ttgacggcgg caccttccag 480

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atgggcgaac gttttggtgg tcagatcctc gataccgttg atatcgaaaa ctacatttct 780

gtaccgaaga ccgaaggcca gaaactggca ggtgcgctga aagttcatgt tgatgaatac 840

gacgttgatg tgatcgacag ccagagcgcg agcaaactga tcccggcagc ggttgaaggc 900

ggcctgcatc agattgaaac agcttctggc gcggtactga aagcacgcag cattatcgtg 960

gcgactggtg caaaatggcg caacatgaac gttcctggcg aagatcagta tcgcaccaaa 1020

ggcgtgacct actgcccgca ctgcgacggc ccgctgttta aaggcaaacg cgtagcggtt 1080

atcggcggcg gtaactccgg cgtggaagcg gcaattgacc tggcgggtat cgttgagcac 1140

gtaacgctgc tggaatttgc gccagaaatg aaagccgacc aggttctgca ggacaaacta 1200

cgcagcctga aaaacgtcga cattattctg aatgcgcaaa ccacggaagt gaaaggcgac 1260

ggtagcaaag tcgtaggtct ggaatatcgc gatcgtgtca gcggcaatat tcacaacatc 1320

gaactggccg gtattttcgt ccagattggt ctgctgccga acaccaactg gctggaaggc 1380

gcagtcgaac gtaatcgcat gggcgagatt atcattgatg cgaaatgcga aaccaacgtc 1440

aaaggcgtgt tcgcagcggg tgactgtacg acggttccgt acaagcagat catcatcg 1498

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aatgcaat 8

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aattaatgtg agttagctca ctcattaggc accccaggct tgacaattaa tcatcggctc 60

gtataatgtg tggaattgtg agcggataac aat 93

<210> 7

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<213> rrnB terminator(Artificial Sequence)

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caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttat 47

Claims

1. A method of identifying insulating sites in a bacterial genome, which are regions within the genome where the level of transcription is low and does not affect or does not affect little the expression of surrounding genes, the method comprising the steps of: and (3) determining that the two ends of the site to be detected have strong transcription terminators by a biological method, and the ends of the two strong transcription terminators are opposite, namely identifying the site as an insulating site.

2. The method for identifying an insulating site in a bacterial genome according to claim 1, wherein: the bacteria are Escherichia coli, lactococcus lactis, Bifidobacterium, Bacteroides fragilis, Bacillus coagulans or Streptococcus thermophilus.

3. The method for identifying an insulating site in a bacterial genome according to claim 2, wherein: the bacterium is escherichia coli, and the insulating site is a sequence located between uspG and aphF genes in the escherichia coli.

4. The method for identifying an insulating site in a bacterial genome according to claim 1, wherein: the sequence corresponding to the insulated site is transcribed into mRNA secondary structure which is consistent with the characteristics of a transcription terminator.

5. A method for preparing recombinant bacteria is characterized in that exogenous genes are inserted into insulating sites of bacteria, so that the expression of the exogenous genes does not influence the expression of other genes of the bacteria; the insulated site is a site which is provided with strong transcription terminators at two ends and the tail ends of the two strong transcription terminators are opposite.

6. The method for producing a recombinant bacterium according to claim 5, wherein: the bacteria are Escherichia coli, lactococcus lactis, Bifidobacterium, Bacteroides fragilis, Bacillus coagulans or Streptococcus thermophilus.

7. The method for producing a recombinant bacterium according to claim 6, wherein: the bacterium is escherichia coli, and the insulating site is a sequence located between uspG and aphF genes in the escherichia coli.

8. The method for producing a recombinant bacterium according to any one of claims 5 to 7, wherein: the introduction mode of the exogenous gene is selected from homologous recombination exchange, CRISPR gene editing and phage transduction.

9. A recombinant bacterium comprising the insulating site according to claim 5 and a foreign gene inserted into the insulating site.

10. Use of the method of claim 1 for identifying insulating sites in a bacterial genome in a bacterial gene editing technique.