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

 
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             <div class="centers">
 
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                 <div class="fb_72">
 
                 <div class="fb_72">
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                     <b>Project Design</b>
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                     <b>Design</b>
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                </div>
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                <div class="fb_48">
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                    <b>Overview</b>
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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 BL21</i> so that it produces phage resistant
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                    protein when being attacked by phage.<br/>
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                    What if it cannot effectively resist? It can explode in vivo before the progeny phages begin to
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                    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/>
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                    Two parallel circuits operate simultaneously. When T4 phage infects <i>E. coli BL21</i> for 5 minutes, the
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                    bacteria can express the resistant protein and emit green fluorescence at the same time. If the
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                    resistant protein successfully defeats T4 phage, the invasion of the phage fails. If the resistance
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                    gene we currently use is not effective against the T4 phage, the phage will continue to infect. When
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                    T4 phage infects E. coli BL21 for 20 minutes, it will trigger the suicide mechanism of the bacteria,
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                    and the bacteria can emit red fluorescence. So we can achieve absolute resistance to phages.<br/>
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                    <br/>
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                    How can the system we developed practically work? This engineered <i>E.coli BL21</i>, is equipped with the
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                    promoters that can be switched on when infected by phages and a library of bacteriophage resistant
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                    proteins so it can flexibly resist and report phages.
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                    <!--                View more-->
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                    <a href="#phage" style="text-decoration: none">
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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>
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                <b>Phage-resistant Genes</b>
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            <img src="https://static.igem.org/mediawiki/2019/8/89/T--JiangnanU_China--project_designs_0.png"
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             <br />
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             <div class="split_small"></div>
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             <div class="fm_22">
 
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                 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
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                 Recombinant <i>E. coli</i> resistant to phage infection will be constructed. It will be mainly divided into four
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                phage-resistant genes in the genome of e. coli, which can be obtained by PCR from existing bacteria.
+
                 parts.
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                <i>AbpA</i> and <i>AbpB</i> impaired the synthesis of late gene of phage transcripts, which resulted in poor
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                expression of late proteins and consequently no phage propagation. By the way, endogenous or exogenous
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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
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                plasmid that links <i>abpA</i> and <i>abpB</i> at the same time, and then use IPTG to induce the expression of
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                resistant proteins.<br/>
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                The plasmid equipped with <i>abpA</i> and <i>abpB</i> , however, can only reduce the susceptibility of bacteria to T4
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                phage as our experiment had showed, but do not have complete resistance. We intend to screen a T4
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                phage-resistant gene by ourselves, which is an innovative and bold idea!<br/>
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                We used ARTP, phage as a stimulus, to get 8 T4 phage-resistant mutants and performed whole-genome
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                sequencing on the strains finally obtained. By analyzing the result, we found the corresponding
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                sequences of T4 phage-resistant proteins that might be produced in the mutants.<br/>
+
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                From the sequencing results, we found that 4 of the genes (<i>rzpD, gntR, yhjH, nuoE</i>) in the genome of the
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                mutant may be related to the resistance. By constructing plasmids to verify their resistance to the T4
+
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                 phage, we finally selected a suitable protein, and the corresponding sequence is our new resistance gene
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                (<i>antP</i>).
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             </div>
 
             <div class="split_small"></div>
 
             <div class="split_small"></div>
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             <img src="https://static.igem.org/mediawiki/2019/2/29/T--JiangnanU_China--project_design_0.png"
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                alt="Phage-resistant Genes">
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                1. Looking for Inducible Promoters
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            </div>
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            <br/>
 
             <div class="fm_22">
 
             <div class="fm_22">
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                 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 will be measured to find parts that
 +
                can respond to phage infection at latent period and burst period. Fluorescence gene <i>gfp</i> and
 +
                <i>mCherry</i>
 +
                will be used to characterize them.
 
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             <img src="https://static.igem.org/mediawiki/2019/d/db/T--JiangnanU_China--design3.png"
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                 alt="Phage-resistant Genes" style="width: 100%;height: auto">
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             <div class="split_small"></div>
 
             <div class="split_small"></div>
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             <div class="fm_22">
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                 These are the two genetic circuits that we ended up designing.
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                 2. Looking for Anti-phage Protein
 
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            <div class="fb_72"><b>Timed promoter</b></div>
 
 
             <br/>
 
             <br/>
 
             <div class="fm_22">
 
             <div class="fm_22">
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                 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 will search for resistant parts that can resist phage infection through literature, and
−
                 freeze it with liquid nitrogen, and find the needed promoter <i>PputA</i> and <i>PglcF</i> through transcriptional
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                use plate test to determine the resistance effect of the parts.
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                 analysis.
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                <br/><br/>
 +
 +
                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.
 
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             <img src="https://static.igem.org/mediawiki/2019/f/f3/T--JiangnanU_China--project_designs_2.png"
 
                 style="width: 100%;height: auto;">
 
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             <div class="fm_22">
 
             <div class="fm_22">
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                 The <i>PputA</i> gene was expressed 5 minutes after phage infection, while the <i>PglcF</i> gene was not expressed
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                 Finally, we will obtain some mutant strains and we will select key sites by comparing the whole genome.
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                until 20 minutes after infection (no expression at 5 minutes).
+
         
 
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             <div class="split_small"></div>
 
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                 style="width: 100%;height: auto;">
 
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             <div class="split_small"></div>
 
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             <div class="fm_22">
 
             <div class="fm_22">
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                 We used the fluorescent protein gene and the found promoter to construct two plasmids to introduce into
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                 Anti-phage detection is carried out on the selected anti-phage part, and the part with the best
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                 <i>E∙coli</i>, and used phage infection to verify whether the promoter was what we wanted.
+
                 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.
 
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            <div class="fb_48">
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                3. Kill Switch
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            </div>
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            <br/>
 +
            <div class="fm_22">
 +
                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.
 +
            </div>
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            <div class="split_small"></div>
 +
             <img src="https://static.igem.org/mediawiki/2019/c/cc/T--JiangnanU_China--project_designs_4.png"
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                4. Application
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            <br/>
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            <div class="fm_22">
 +
                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.
 +
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Latest revision as of 19:01, 21 October 2019

JiangNan

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.
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.
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