Team:JiangnanU China/Design
Project Design
Overview
This year, our team JiangnanU_China dedicated to address phage infection and subsequently
yield-loosing issue in fermentation industry part by our innovative genetically engineered bacteria.
Based on our design, our team genetically modify E. coli BL21 so that it produces phage resistant
protein when being attacked by phage.
What if it cannot effectively resist? It can explode in vivo before the progeny phages begin to assemble. We can not only protect the surrounding bacteria from infection, but also make the detection personnel more intuitive and convenient to detect the invasion of phages by fluorescence, saving time and timely stop loss.
Two parallel circuits operate simultaneously. When T4 phage infects E. coli BL21 for 5 minutes, the bacteria can express the resistant protein and emit green fluorescence at the same time. If the resistant protein successfully defeats T4 phage, the invasion of the phage fails. If the resistance gene we currently use is not effective against the T4 phage, the phage will continue to infect. When T4 phage infects E.coli BL21 for 20 minutes, it will trigger the suicide mechanism of the bacteria, and the bacteria can emit red fluorescence. So we can achieve absolute resistance to phages.
How can the system we developed practically work? This engineered E.coli BL21, is equipped with the promoters that can be switched on when infected by phages and a library of bacteriophage resistant proteins so it can flexibly resist and report phages.
What if it cannot effectively resist? It can explode in vivo before the progeny phages begin to assemble. We can not only protect the surrounding bacteria from infection, but also make the detection personnel more intuitive and convenient to detect the invasion of phages by fluorescence, saving time and timely stop loss.
Two parallel circuits operate simultaneously. When T4 phage infects E. coli BL21 for 5 minutes, the bacteria can express the resistant protein and emit green fluorescence at the same time. If the resistant protein successfully defeats T4 phage, the invasion of the phage fails. If the resistance gene we currently use is not effective against the T4 phage, the phage will continue to infect. When T4 phage infects E.coli BL21 for 20 minutes, it will trigger the suicide mechanism of the bacteria, and the bacteria can emit red fluorescence. So we can achieve absolute resistance to phages.
How can the system we developed practically work? This engineered E.coli BL21, is equipped with the promoters that can be switched on when infected by phages and a library of bacteriophage resistant proteins so it can flexibly resist and report phages.
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Phage-resistant Genes
In the early stage, we found abpA and abpB after consulting the literature. abpA and abpB are two
phage-resistant genes in the genome of e. coli, which can be obtained by PCR from existing bacteria.
AbpA and AbpB impaired the synthesis of late gene of phage transcripts, which resulted in poor
expression of late proteins and consequently no phage propagation. By the way, endogenous or exogenous
AbpA and AbpB had no effect on bacterial growth and bacterial DNA synthesis. We intend to construct a
plasmid that links abpA and abpB at the same time, and then use IPTG to induce the expression of
resistant proteins.
The plasmid equipped with abpA and abpB , however, can only reduce the susceptibility of bacteria to T4 phage as our experiment had showed, but do not have complete resistance. We intend to screen a T4 phage-resistant gene by ourselves, which is an innovative and bold idea!
We used ARTP, phage as a stimulus, to get 8 T4 phage-resistant mutants and performed whole-genome sequencing on the strains finally obtained. By analyzing the result, we found the corresponding sequences of T4 phage-resistant proteins that might be produced in the mutants.
From the sequencing results, we found that 4 of the genes (rzpD, gntR, yhjH, nuoE) in the genome of the mutant may be related to the resistance. By constructing plasmids to verify their resistance to the T4 phage, we finally selected a suitable protein, and the corresponding sequence is our new resistance gene (antP).
The plasmid equipped with abpA and abpB , however, can only reduce the susceptibility of bacteria to T4 phage as our experiment had showed, but do not have complete resistance. We intend to screen a T4 phage-resistant gene by ourselves, which is an innovative and bold idea!
We used ARTP, phage as a stimulus, to get 8 T4 phage-resistant mutants and performed whole-genome sequencing on the strains finally obtained. By analyzing the result, we found the corresponding sequences of T4 phage-resistant proteins that might be produced in the mutants.
From the sequencing results, we found that 4 of the genes (rzpD, gntR, yhjH, nuoE) in the genome of the mutant may be related to the resistance. By constructing plasmids to verify their resistance to the T4 phage, we finally selected a suitable protein, and the corresponding sequence is our new resistance gene (antP).
We constructed a plasmid that connects antP (antP1) and abPAB (antP2) and expected it to perform better.
These are the two genetic circuits that we ended up designing.
Timed promoter
To find the needed promoter, we will infect the E. coli with phage for 0min, 5min and 20min and then
freeze it with liquid nitrogen, and find the needed promoter PputA and PglcF through transcriptional
analysis.
The PputA gene was expressed 5 minutes after phage infection, while the PglcF gene was not expressed
until 20 minutes after infection (no expression at 5 minutes).
We used the fluorescent protein gene and the found promoter to construct two plasmids to introduce into
E∙coli, and used phage infection to verify whether the promoter was what we wanted.
