Difference between revisions of "Team:JiangnanU China/Demonstrate"

We obtained phage-inducible promoters PputA and PglcF by transcriptomics data, ligated these two different time-responsive inducible promoters to the fluorescent genes gfp and mcherry, respectively, and transformed them into E. coli BL21. The two strains BL21-PputA-gfp and BL21-glcF-mcherry were cultured overnight at 37 ° C for about 12-16 hours, then transferred to fresh LB medium at 1%, cultured for about 2 hours. After that added 1% freshly centrifuged T4 phage solution, and shaken at 37 ° C for 30 min. Then take the 10 μL to make tablets, and fluorescence observation was performed by a laser confocal microscope. At the same time, we set up a mutant strain without phage as a control.

From the results, we can find that the strains E.coli BL21-PputA-gfp and E. coliBL21-PglcF-mcherry without phage showed no signs of fluorescence. The two bacteria infected with phage showed obvious fluorescence. But overall, the number of bacteria that show fluorescence was not that much, because not every bacterium was infected with T4 phage. This indicated that the promoters we selected is silent in the absence of phage infestation and does not affect the expression of subsequent proteins.

We found a resistance protein, AbpAB, from the literature, and found that the protein only reduced phage sensitivity. To obtain a strain that is fully immunized against the T4 phage, we mutated and sequenced E. coli BL21 to obtain four key anti-phage-related genes gntR, rzpD, yhjH, nuoE.

Overexpression of these four genes, further experimental results show that gntR has the best anti-reverse effect.

We connect gntR with abpAB on pET28a, and transformed them into E. coli BL21. We co-expressed abpAB and gntR, and surprisingly obtained a recombinant strain that is completely immune to phage.

In the case of determining that the combination resistance protein is effective, we chose the constitutive promoter PrsmH to constitutively express the resistance protein in an attempt to verify whether the resistance protein has an effect on cell growth.

We ligated the combined resistance protein gene and the kill switch P-1 (P-1 part链接)after the latent phage-inducible promoter and the burst-inducible promoter, and performed the phage infection assay on the LB agar plate, and got very good resistance.

In addition, we inoculated E. coli BL21 and recombinant E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 in LB liquid medium to raise the logarithmic growth phase, ie OD 0.6-0.8 . Than the fresh phage solution was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found that the recombinant grew well.

We cooperated with NINGXIA EPPEN BIOTECH CO.,LTD to carry out small-scale and pilot test fermentation experiments of resistant strain in the special fermentation laboratory of Jiangnan University. We transferred the constructed plasmid into a production strain producing γ-aminobutyric acid, β-aminobutyric acid, 2, 5-dimethyl pyrazine to obtain a resistant strain for producing a specific product.
The fermentation laboratory is subjected to UV irradiation and ozone fumigation prior to formal fermentation to remove phage that may be present. The resistant strain and the control were added to 1% phage about 6-8 hours after inoculation, and the fermentation was continued for 15 hours. During the fermentation, the OD and product concentration of the fermentation were measured, and the effects of the phage on the two were observed.