Protocol

<1. Strains and the plasmids

Lactobacillus plantarum ZJ316 was purchased from Zhili Zhongte Biological Technology Co. (Wuhan,China). H. pylori ATCC43504 strain was purchased from Crisprbio Biological Technology Co. (Qingdao,China).The vector pUC-19 was purchased from Zhili Zhongte Biological Technology Co. (Wuhan,China).The BL21 Competent cell was purchased from Shanghai weidibio Biological Technology Co.(Shanghai,China)

<2. Chemicals and Materials

LB liquid culture medium in bags (dry powder) was purchased from coolaber Biological Technology Co.（Beijing,China）.Bacteria Genomic DNA Kit was purchased from Trans

Gene Biological Technology Co.(Beijing,China). T4 DNA Ligase was purchased from Vazyme

Biological Technology Co.（Nanjing,China）. Instant Error-prone PCR Kit was purchased from Tiandz Biological Technology Co.（Beijing,China）. PowerPol 2x PCR Mix with Dye was purchased from ABclonal Biological Technology Co.（Wuhan,China）.Kannamycin Sulfate and X-gal was purchased from coolaber Biological Technology Co. （Beijing,China）FastDigest Xhol EcorI and Xbal was purchased from Thermo Fisher Biological Technology Co.（Shanghai,China）

<3. Constructing mutant library

Select two genes that respectively control the synthesis of peptide chains at both ends of bacteriocin, extract bacterial genes, conduct error prone PCR, obtain a large number of mutations, and then randomly recombine the mutant genes through bacterial conjugation, so as to build a mutation library.

<4. Cloning bacteriocin encoding genes

The bacteria were lysed by enzymolysis, and DNA was specifically adsorbed by silica gel membrane centrifuge column. Since the bacteria were gram-positive, additional lysozyme was needed. The genomic DNA of bacteria was obtained.

<5.Resistance screening

The plasmid carries the ampicillin resistant gene specifically expressed. Adding ampicillin to the culture medium can screen the E. coli successfully transferred into the plasmid, and the plasmid successfully connected with the gene can be screened through blue and white spots. On this basis, the Escherichia coli that successfully transferred the mutant gene into the body was preliminarily screened.

<6.Gene recombination

Cultivate the two strains carrying the mutant gene together for a period of time, so that they can produce bacterial conjugation and genetic recombination. It is possible to integrate the two mutant genes into the genome of one strain,

<7. Rescreening

Incubate Helicobacter pylori on Columbia blood plate, culture the bacteria joined to obtain culture products respectively, pour the culture products on Helicobacter pylori, detect the bacteriocin strength of the strain, and screen out the strain with strong bacteriocin.

<8. Mutation sites analysis

Carry out gene detection on the screened strains and analyze the mutation.