Team:JiangnanU China/Design
Recombinant E. coli resistant to phage infection was constructed. It is mainly divided into four
parts.
1. Looking for Inducible Promoters
Therefore, transcriptome data from different stages of phage infection were measured to find parts that
could respond to phage infection at latent period and burst period Fluorescence gene gfp and
mCherry
were used to characterize them.
2. Looking for Anti-phage Protein
On the one hand, we searched for resistant parts that can resist phage infection through literature, and
used plate test to determine the resistance effect of the parts.
On the other hand, we use ARTP (Atmospheric and Room Temperature Plasma) mutagenesis screening to screen for bacteriophage-resistant parts. Specifically, we identified the mutant strain by co-culture with the phage, and after sorting out the mutant strain, we cultured all the mutant strains for ten generations to strengthen the mutant sites. In this process, the phage plate test has been carried out to eliminate the degraded resistant strains.
On the other hand, we use ARTP (Atmospheric and Room Temperature Plasma) mutagenesis screening to screen for bacteriophage-resistant parts. Specifically, we identified the mutant strain by co-culture with the phage, and after sorting out the mutant strain, we cultured all the mutant strains for ten generations to strengthen the mutant sites. In this process, the phage plate test has been carried out to eliminate the degraded resistant strains.
Finally, four phage-resistant mutant strains were obtained. By comparing the whole genome, we selected
key sites.
Anti-phage detection was carried out on the selected anti-phage part, and the part with the best
anti-phage effect was cascaded with the anti-phage part screened in the literature, and both of them
were connected to the inducible promoter that could respond to phages in the latent period.
3. Kill Switch
In the second part, we were to find anti-phage parts which could in the latent period resist to phage.
However, if the phage skip our first line of defense, we were able to ligate the kill switch with the
burst period inducible promoter to kill the cell before the complete assembly of phage.
4. Application
The constructed recombinant E. coli BL21 is applied to produce γ-aminobutyric acid in 5 L and 30 L
fermentation cultures in the laboratory.
