Team:JiangnanU China/Design
Recombinant E. coli resistant to phage infection will be constructed. It will be mainly divided into four
parts.
1. Looking for Inducible Promoters
Therefore, transcriptome data from different stages of phage infection will be measured to find parts that
can respond to phage infection at latent period and burst period. Fluorescence gene gfp and
mCherry
will be used to characterize them.
2. Looking for Anti-phage Protein
On the one hand, we will search for resistant parts that can resist phage infection through literature, and
use plate test to determine the resistance effect of the parts.
On the other hand, we will use ARTP (Atmospheric and Room Temperature Plasma) mutagenesis screening to screen for bacteriophage-resistant parts. Specifically, we identify the mutant strain by co-culture with the phage, and after sorting out the mutant strain, we culture 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 will use ARTP (Atmospheric and Room Temperature Plasma) mutagenesis screening to screen for bacteriophage-resistant parts. Specifically, we identify the mutant strain by co-culture with the phage, and after sorting out the mutant strain, we culture 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, we will obtain some mutant strains and we will select key sites by comparing the whole genome.
Anti-phage detection is carried out on the selected anti-phage part, and the part with the best
anti-phage effect is cascaded with the anti-phage part screened in the literature, and both of them
are connected to the inducible promoter that could respond to phages in the latent period.
3. Kill Switch
In the second part, we plan 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 are able to ligate the kill switch with the
burst period inducible promoter to kill the cell before the complete assembly of phage.
4. Application
When the recombinant mutant is constructed, we will revisit our original purpose, which is to play a role in the practical application. In view of the advantages of our school Jiangnan University in fermentation engineering, we will apply our construction circuit to the production strain to verify its ability to resist phage. This experiment will be done in a specific laboratory where our school works with the respective companies, and we will ensure the safety of the entire experiment and prevent any bacteria and phage from leaking.
