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

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                     This year, our team JiangnanU_China dedicated to address phage infection and subsequently
 
                     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.
 
                     yield-loosing issue in fermentation industry part by our innovative genetically engineered bacteria.
                     Based on our design, our team genetically modify <i>E. coli BL21</i> so that it produces phage resistant
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                     Based on our design, our team genetically modify <i>E. coli </i>BL21 so that it produces phage resistant
                     protein when being attacked by phage.<br/>
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                     protein when being attacked by phage.<br/><br/>
 
                     What if it cannot effectively resist? It can explode in vivo before the progeny phages begin to
 
                     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
 
                     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,
 
                     detection personnel more intuitive and convenient to detect the invasion of phages by fluorescence,
                     saving time and timely stop loss.<br/>
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                     saving time and timely stop loss.<br/><br/>
                     Two parallel circuits operate simultaneously. When T4 phage infects <i>E. coli BL21</i> for 5 minutes, the
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                     Two parallel circuits operate simultaneously. When T4 phage infects <i>E. coli</i> BL21 for 5 minutes, the
 
                     bacteria can express the resistant protein and emit green fluorescence at the same time. If 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
 
                     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
 
                     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,
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                     T4 phage infects <i>E.coli</i> 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.<br/>
 
                     and the bacteria can emit red fluorescence. So we can achieve absolute resistance to phages.<br/>
                     <br/>
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                     <br/><br/>
                     How can the system we developed practically work? This engineered <i>E.coli BL21</i>, is equipped with the
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                     How can the system we developed practically work? This engineered <i>E.coli</i> BL21, is equipped with the
 
                     promoters that can be switched on when infected by phages and a library of bacteriophage resistant
 
                     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.
 
                     proteins so it can flexibly resist and report phages.
 
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                 <i>AbpA</i> and <i>AbpB</i> had no effect on bacterial growth and bacterial DNA synthesis. We intend to construct a
 
                 <i>AbpA</i> and <i>AbpB</i> had no effect on bacterial growth and bacterial DNA synthesis. We intend to construct a
 
                 plasmid that links <i>abpA</i> and <i>abpB</i> at the same time, and then use IPTG to induce the expression of
 
                 plasmid that links <i>abpA</i> and <i>abpB</i> at the same time, and then use IPTG to induce the expression of
                 resistant proteins.<br/>
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                 resistant proteins.<br/><br/>
 
                 The plasmid equipped with <i>abpA</i> and <i>abpB</i> , however, can only reduce the susceptibility of bacteria to T4
 
                 The plasmid equipped with <i>abpA</i> and <i>abpB</i> , 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 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!<br/>
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                 phage-resistant gene by ourselves, which is an innovative and bold idea!<br/><br/>
 
                 We used ARTP, phage as a stimulus, to get 8 T4 phage-resistant mutants and performed whole-genome
 
                 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
 
                 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.<br/>
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                 sequences of T4 phage-resistant proteins that might be produced in the mutants.<br/><br/>
 
                 From the sequencing results, we found that 4 of the genes (<i>rzpD, gntR, yhjH, nuoE</i>) in the genome of the
 
                 From the sequencing results, we found that 4 of the genes (<i>rzpD, gntR, yhjH, nuoE</i>) in the genome of the
 
                 mutant may be related to the resistance. By constructing plasmids to verify their resistance to the T4
 
                 mutant may be related to the resistance. By constructing plasmids to verify their resistance to the T4

Revision as of 06:16, 21 October 2019

JiangNan

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).
Phage-resistant Genes
We constructed a plasmid that connects antP (antP1) and abPAB (antP2) and expected it to perform better.
Phage-resistant Genes
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.
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