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

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.

According to the advice of corporate stakeholders, if the Genetic Modified (GM) strain is to be applied in industry, our components cannot have a great influence on bacterial growth. Since it is not possible to directly see from the figure which element has the least influence on the growth of the bacteria, we use the Grey Relation Analysis（GRA） method to analyze the growth curve of the bacteria connecting the various components. We used the Entropy Weight Method (EWM) method to determine the weight of each growth point to select the most similarly modified strain (the highest correlation), which is the component that has the least impact on bacterial growth. At the same time, after consulting the industry experts, we revised the weights according to the experts' recommendations to evaluate the components again, making them more in line with the real situation of production, that is, the most suitable components for industrial production. Using GRA's two evaluations of the four components at different weights, we selected the component gntR to cascade abpAB to construct a stronger resistant strain for better industrial performance.

In the case of determining that the combination resistance protein is effective, we choose the constitutive promoter P_rsmH to constitutively express the resistance protein in an attempt to verify whether the resistance protein has an effect on cell growth. First we linked this constitutive promoter to gfp, demonstrating the validity of this constitutive promoter. The effect of overexpression of the resistance protein AbpAB on growth was then verified. The results showed that P_rsmH had a good effect, and overexpression of AbpAB had no significant effect on the growth of E. coli BL21.

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. This ensures that our experiments are controllable without any phage and engineered bacteria leaking.
We transferred the constructed plasmid into a production strain producing γ-aminobutyric acid (GABA).
The fermentation laboratory is subjected to UV irradiation and ozone fumigation prior to formal fermentation to remove phage that may be present. TheE. coli BL21-pET28a-P_putA-abpAB-gntR-P_glcF-P-1, E. coli BL21-pET28a-P_putA-abpAB and the control (E. coli BL21) were added to 1% T4 phage about 6 hours after inoculation, and the fermentation was continued for 10 hours. During the fermentation, the OD600 was measured, and the effects of the phage on the three were observed.

Then we used whole-cell transformation with a combination of resistant strain E. coli BL21-pET28a-P_putA-abpAB-gntR-P_glcF-P-1 and we got a good whole-cell transformation ability of the resistant strain (Figure 13).

From the results, our resistant composite part has great advantages in the production of γ-aminobutyric acid and are not threatened by T4 phage. The productivity of γ-aminobutyric acid is 278.3 g/L, and the molar conversion rate is really high, reaching 98.4%, which means that the circuit we built can be used in production without any impact (Table 1).