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

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     <style>
 
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             background-image: url('https://static.igem.org/mediawiki/2019/9/94/T--JiangnanU_China--project_design_bgd.png');
 
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<body>
 
<body>
 
<div class="bgd" id="head">
 
<div class="bgd" id="head">
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    <div class="split_small"></div>
 
     <div class="split"></div>
 
     <div class="split"></div>
 
     <div class="contents" style="color: white">
 
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             <div class="centers">
 
             <div class="centers">
 
                 <div class="fb_72">
 
                 <div class="fb_72">
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                     <b>Project Design</b>
+
                     <b>Design</b>
 
                 </div>
 
                 </div>
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                <div class="fb_48">
 
−
                    <b>Overview</b>
 
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                </div>
 
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                <br/>
 
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                <div class="fm_22" style="font-size: 1em">
 
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                    This year, our team JiangnanU_China dedicated to address phage infection and subsequently
 
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                    yield-loosing issue in fermentation industry part by our innovative genetically engineered bacteria.
 
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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/><br/>
 
−
                    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
 
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                    detection personnel more intuitive and convenient to detect the invasion of phages by fluorescence,
 
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                    saving time and timely stop loss.<br/><br/>
 
−
                    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
 
−
                    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 <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/>
 
−
                    <br/><br/>
 
−
                    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
 
−
                    proteins so it can flexibly resist and report phages.
 
−
                </div>
 
−
 
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                <div class="split"></div>
 
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                <!--                View more-->
 
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                <a href="#phage" style="text-decoration: none">
 
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                    <div class="row" style="align-content: center;color: white;">
 
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                        <img
 
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                                src="https://static.igem.org/mediawiki/2019/0/09/T--JiangnanU_China--host_liubianxing.png"
 
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                                alt="back" style="width: 6%;height:6%;">
 
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                        <div class="fb_48" style="margin-left: 2%;margin-top: 1%">View all</div>
 
−
                    </div>
 
−
                </a>
 
−
 
−
 
 
             </div>
 
             </div>
 
         </div>
 
         </div>
 
     </div>
 
     </div>
 
</div>
 
</div>
−
 
−
 
−
 
  
  
 
<div class="split"></div>
 
<div class="split"></div>
−
<div class="contents">
+
<div class="contents" id="phage">
 
     <div class="column">
 
     <div class="column">
 
         <div class="centers">
 
         <div class="centers">
−
             <div class="fb_72">
+
 
−
                <b>Phage-resistant Genes</b>
+
             <div class="split_small"></div>
−
             </div>
+
             <img src="https://static.igem.org/mediawiki/2019/8/89/T--JiangnanU_China--project_designs_0.png"
−
             <br />
+
                style="width: 100%;height: auto;">
 +
             <div class="split_small"></div>
 
             <div class="fm_22">
 
             <div class="fm_22">
−
                 In the early stage, we found <i>abpA</i> and <i>abpB</i> after consulting the literature. <i>abpA</i> and <i>abpB</i> are two
+
                 Recombinant <i>E. coli</i> resistant to phage infection was constructed. It is mainly divided into four
−
                phage-resistant genes in the genome of e. coli, which can be obtained by PCR from existing bacteria.
+
                 parts.
−
                <i>AbpA</i> and <i>AbpB</i> 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
+
−
                <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
+
−
                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
+
−
                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/><br/>
+
−
                 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.<br/><br/>
+
−
                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
+
−
                phage, we finally selected a suitable protein, and the corresponding sequence is our new resistance gene
+
−
                (<i>antP</i>).
+
 
             </div>
 
             </div>
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
−
             <img src="https://static.igem.org/mediawiki/2019/2/29/T--JiangnanU_China--project_design_0.png"
+
             <div class="fb_48">
−
                alt="Phage-resistant Genes">
+
                First Part-looking for Inducible Promoters
 +
            </div>
 +
            <br/>
 
             <div class="fm_22">
 
             <div class="fm_22">
−
                 We constructed a plasmid that connects <i>antP (antP1)</i> and <i>abpAB (antP2)</i> and expected it to perform better.
+
                 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 <i>gfp</i> and
 +
                <i>mCherry</i>
 +
                were used to characterize them.
 
             </div>
 
             </div>
−
 
+
            <div class="split_small"></div>
−
 
+
             <img src="https://static.igem.org/mediawiki/2019/0/04/T--JiangnanU_China--project_designs_1.png"
−
 
+
−
          <div class="split_small"></div>
+
−
             <img src="https://static.igem.org/mediawiki/2019/a/a3/T--JiangnanU_China--project_demonstrate_0_%282%29.png"
+
 
                 style="width: 100%;height: auto;">
 
                 style="width: 100%;height: auto;">
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
−
 
−
 
  
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
−
            <img src="https://static.igem.org/mediawiki/2019/8/8d/T--JiangnanU_China--project_demonstrate_13.png"
+
             <div class="fb_48">
−
                alt="Phage-resistant Genes" style="width: 100%;height: auto">
+
                 Second Part-looking for Anti-phage Protein
−
             <div class="split_small"></div>
+
−
 
+
−
            <div class="fm_22">
+
−
                 These are the two genetic circuits that we ended up designing.
+
 
             </div>
 
             </div>
−
            <div class="split"></div>
 
−
            <div class="fb_72"><b>Timed promoter</b></div>
 
 
             <br/>
 
             <br/>
 
             <div class="fm_22">
 
             <div class="fm_22">
−
                 To find the needed promoter, we will infect the <i>E. coli</i> with phage for 0min, 5min and 20min and then
+
                 On the one hand, we searched for resistant parts that can resist phage infection through literature, and
−
                 freeze it with liquid nitrogen, and find the needed promoter P<i>putA</i> and P<i>glcF</i> through transcriptional
+
                used plate test to determine the resistance effect of the parts.
−
                 analysis.
+
                <br/>
 +
                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.
 
             </div>
 
             </div>
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
−
             <img src="https://static.igem.org/mediawiki/2019/6/61/T--JiangnanU_China--project_design_1.png"
+
             <img src="https://static.igem.org/mediawiki/2019/f/f3/T--JiangnanU_China--project_designs_2.png"
 
                 style="width: 100%;height: auto;">
 
                 style="width: 100%;height: auto;">
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
 
             <div class="fm_22">
 
             <div class="fm_22">
−
                 The P<i>putA</i> gene was expressed 5 minutes after phage infection, while the P<i>glcF</i> gene was not expressed
+
                 Finally, four phage-resistant mutant strains were obtained. By comparing the whole genome, we selected
−
                 until 20 minutes after infection (no expression at 5 minutes).
+
                 key sites.
 
             </div>
 
             </div>
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
−
             <img src="https://static.igem.org/mediawiki/2019/a/a5/T--JiangnanU_China--project_design_2.png"
+
             <img src="https://static.igem.org/mediawiki/2019/2/27/T--JiangnanU_China--project_designs_3.png"
 
                 style="width: 100%;height: auto;">
 
                 style="width: 100%;height: auto;">
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
 
             <div class="fm_22">
 
             <div class="fm_22">
−
                 We used the fluorescent protein gene and the found promoter to construct two plasmids to introduce into
+
                 Anti-phage detection was carried out on the selected anti-phage part, and the part with the best
−
                 <i>E∙coli</i>, and used phage infection to verify whether the promoter was what we wanted.
+
                 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.
 
             </div>
 
             </div>
 +
 +
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
−
             <img src="https://static.igem.org/mediawiki/2019/c/c6/T--JiangnanU_China--project_design_3.png" style="width: 100%;height: auto">
+
            <div class="fb_48">
 +
                Third Part-kill Switch
 +
            </div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                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 ligated the kill switch to the
 +
                burst period inducible promoter, killing the cell before the complete assembly of phage.
 +
            </div>
 +
            <div class="split_small"></div>
 +
             <img src="https://static.igem.org/mediawiki/2019/c/cc/T--JiangnanU_China--project_designs_4.png"
 +
                style="width: 100%;height: auto;">
 +
            <div class="split_small"></div>
 +
 
 +
            <div class="split_small"></div>
 +
            <div class="fb_48">
 +
                Four Part-application
 +
            </div>
 +
            <br/>
 +
            <div class="fm_22">
 +
                The constructed recombinant <i>E. coli</i> is applied to produce γ-aminobutyric acid in 5 L and 30 L
 +
                fermentation cultures in the laboratory.
 +
            </div>
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/c/c8/T--JiangnanU_China--project_designs_6.png"
 +
                style="width: 100%;height: auto;">
 +
            <div class="split_small"></div>
 +
 
 +
 
 +
            <!--            书签-->
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
 
             <a href="#head"><img src="https://static.igem.org/mediawiki/2019/2/24/T--JiangnanU_China--host_back.png"
 
             <a href="#head"><img src="https://static.igem.org/mediawiki/2019/2/24/T--JiangnanU_China--host_back.png"

Revision as of 06:45, 21 October 2019

JiangNan

Recombinant E. coli resistant to phage infection was constructed. It is mainly divided into four parts.
First Part-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.
Second Part-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.
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.
Third Part-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 ligated the kill switch to the burst period inducible promoter, killing the cell before the complete assembly of phage.
Four Part-application

The constructed recombinant E. coli is applied to produce γ-aminobutyric acid in 5 L and 30 L fermentation cultures in the laboratory.
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