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

We added 1 μL of phage-infected fermentation broth to a plate containing E. coli BL21.
After proper culture for a period of time, plaque appeared on the plate (Fig.1.)
We isolated the phages from the plate and photographed them using a projective electron microscope (Fig. 2).
We finally determined that the T4 phages infected our fermentation broth by sequencing the genome of the phages.

In order to screen inducible promoters, we first made the one-step growth curve of phages (Fig. 3). After that, we selected two time points of phage infection for 5min （in the latent period of phage infection）and phage infection for 20min （in the burst period of phage infection）through the one-step growth curve of phage. By analyzing transcriptome data，we selected inducible promoter PputA (Fig.4) for 5 min and inducible promoter PglcF (Fig.5) for 20 min.

In E. coli BL21, we connected the green fluorescence gene gfp with the inducible promoter PputA for 5 min (Fig.6) and the red fluorescence gene mCherry with the inducible promoter PglcF for 20 min (Fig.7) in our genetic circuits. After infecting the bacteria with phages for the corresponding time, we observed that the infected cells gave off green and red fluorescence respectively.

When we had components that responded to phage infection, we searched for anti-phage parts through literature search and mutagenesis screening.

3.1 Anti-phage part from literature
Through literature search, we found a resistant protein AbpAB that can resist T4 phage. Protein AbpAB can inhibit the replication and late gene expression of phage, which resulted in blocking of phage propagation. AbpAB have no effect on the bacterial growth(Fig8.) which is important to the industry. However, protein AbpAB didn’t work well as we expected(Fig 9.).

3.2. Anti-phage part from mutation screening
To get more efficient phage resistant parts, we used the ARTP(Atmospheric and room temperature plasma) mutagenesis system to obtain a large number of mutant strains. Then we co-cultured the mutant strains with the phage and screened the mutant strains that could resist phage infection. In the screening process, we continuously verified their resistance, eliminated the bacterial strains with degraded resistance and retained the ones with excellent resistance. Then we obtained 4 mutant strains which were resistant to phage and four key mutation sites (nuoE, yhjH, rzpD, and gntR) through comparation of genome (Fig. 10). Resistance tests on these key sites were performed respectively(Fig 11.). 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 component has the least influence on the growth of the bacteria, we use the Grey Relation Analysis（GRA） method to analyze the growth curve（Fig 12.） of the bacteria connecting the various components. We used the Entropy Weight Method (EWM) 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.(Fig 13.)

3.3. Cascade of protein AbpAB and GntR
First, resistant proteins AbpAB and GntR were verified by SDS-PAGE（Fig 14.）Then，We connected gntR with abpAB in pET-28a plasmid , and transformed them into E. coli BL21. We co-expressed abpAB and gntR, and subsequently obtained a recombinant strain that is resistant to phage（Fig 15.）. The result showed that two proteins works better together.

In order to prevent the phage from escaping the attack of our resistant protein, we connected a kill switch p-1 (BBa_K628000) with the 20 min induction promoter(Fig 16).

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, i.e. OD 0.6-0.8 . Then the fresh phage solution was inoculated at the same time and continuous cultured for 1-2 h. As a result, it was found that the recombinant grew well.(Fig 17.).

In addition, we cooperated with Ningxia EPPEN BIOTECH CO.,LTD (http://www.eppen.com.cn) to carry out small-scale and pilot test fermentation experiments of resistant strain in the special fermentation laboratory of Jiangnan University. This ensured that our experiments were controllable without any phage and engineered bacteria leaking.
We transferred the constructed plasmid into an engineering bacteria 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. The E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1, E. coli BL21-pET28a-PputA-abpAB and the control （E. coli BL21） were added T4 phage after six hours of culture, and the fermentation was continued for 10 hours. During the fermentation, the OD 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-PputA-abpAB-gntR-PglcF-P-1, and we got a good whole-cell transformation ability of the resistant strain (Figure 18).
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%(Table 1.), which means that the circuit we built can be used in production without any impact (Figure 19).