Results

<1. Electrophoresis results of PCR products

After the genomic DNA of lactic acid bacteria is extracted, the target gene fragment will be obtained by a common PCR. The PCR products were subjected to agarose gel electrophoresis to obtain fragments of target gene size. The pure target gene can be obtained by cutting the strip where the target gene is located and recycling the glue.

<2. Blue white spot reaction

The characteristics of the plasmid used, if the target gene is successfully transferred into the plasmid, then the enzyme that decomposes the substrate IPTG will not be synthesized, and the colony will be white, on the contrary, blue colony will be formed. Because the plasmid connection will not be 100% successful, the whole plate will be blue and white spots.

<3. Inhibitory effect of mutant strains on helicobacter pylori

By comparing the different inhibitory effects of the cultures of mutant strains on Helicobacter pylori, we can select lactobacillus which can produce stronger bacteriocin.

<4. Sequencing results

Sequencing and analyzing the mutation site to cultivate lactic acid bacteria with stronger inhibitory effect of Helicobacter pylori.

<5. Other model assumptions.

In conventional evolutionary design, we usually evolve a gene to obtain the desired protein. However, through bacterial conjugation, we may try to obtain some evolutionary products under the influence of multiple factors, or explore the interaction mechanism through evolutionary methods.

The dipeptide chain model involved in this paper. If the protein contains multiple peptide chains, the interaction between different peptide chains will greatly affect the function of the whole protein. It should be better to combine the two evolved peptide chains before judging their functions than to combine each peptide chain after evolution alone. The molecular mechanism of protein interaction can be analyzed by analyzing the evolved sites.

Two product model. If one effect can be achieved by both products, then how can they combine and evolve to achieve the best effect? We can use the mutation method that the two products can have different expression levels of the mutation library, then use the principle of bacterial conjugation to make them randomly combine with each other, and finally screen out the best combination. Through this method, some drug matching can be selected. It is also possible to analyze the correlation between the action mechanisms of the two products. If the results of the respective evolution integration and the combination evolution are consistent, or it can be considered that the action mechanisms of the two products are not closely related, so when one drug fails, the other drug may still play a role, which is of some significance for treatment.

Discussion

<1. Result analysis

We did not get ideal results, and speculated that the reasons and updated experimental conditions are as follows:

First. The model selected in the experiment is incorrect. The evolution space of the two peptide chains in this gene may be small, and there may be no better interaction conditions. It is suggested to analyze the protein structure first.Second,The probability of bacterial conjugation in the bacteria is too small to produce a large number of target recombination, or the bacteria can be changed to the species with high conjugation frequency, so that the gene can be fully recombined. Third. It may require special experimental conditions to produce a large number of joints. Fourth. Theoretical speculation may have limitations.

<2. expectation

This technology can be well used in the aspect of orientation technology, and the method of extracting the target gene is very convenient compared with the shotgun method commonly used now. The combination of bacterial conjugation and error prone PCR can better take all the influencing factors into account, and is closer to the experimental goal than one of the indicators, which is more meaningful for the actual production evolution.