Team:JiangnanU China/Demonstrate
Project Demonstrate
Throughout the project, we screened two inducible promoters, mutated to obtain six phage resistance
parts, and found a phage resistance part from the literature, all of which were derived from E.
coli
BL21. We used reporter genes to detect the effectiveness of inducible promoters, and
overexpression
of resistance proteins to verify the effect of proteins on cell growth. We assembled the resistance
parts and promoters to obtain a strong strain that is immune to T4 phage.
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Inducible Promoters PputA And PglcF Can Respond toT4 Phage Infection
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 BL21-PputA-gfp and BL21-glcF-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.
The Effectiveness of T4 Phage Resistance 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.
Constitutive Expression of Resistance Proteins
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.
The Effectiveness of The Overall Gene Circuit
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.
Application of Phage Resistant Strain in Fermentation Growth
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.
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.
