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

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<h1>Design</h1>
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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<h3>What should this page contain?</h3>
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            src: url('https://static.igem.org/mediawiki/2019/4/49/T--JiangnanU_China--FuturaFuturisC-Italic.otf');
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<li>Explanation of the engineering principles your team used in your design</li>
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        }
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<li>Discussion of the design iterations your team went through</li>
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<li>Experimental plan to test your designs</li>
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</ul>
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<h3>Inspiration</h3>
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<ul>
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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</ul>
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            <div class="centers">
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                <div class="fb_72">
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                    <b>Project 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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                </div>
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                <br/>
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                <div class="fm_22" style="font-size: 0.9em">
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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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                </div>
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            </div>
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        </div>
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    </div>
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<div class="split"></div>
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<div class="contents">
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        <div class="centers">
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                <b>Phage-resistant Genes</b>
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            </div>
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            <br />
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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
 +
                phage-resistant genes in the genome of e. coli, which can be obtained by PCR from existing bacteria.
 +
                <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/>
 +
                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/>
 +
                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/>
 +
                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>
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            <div class="split_small"></div>
 +
            <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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            <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.
 +
            </div>
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            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/5/59/T--JiangnanU_China--project_design_4.png"
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                alt="Phage-resistant Genes" style="width: 100%;height: auto">
 +
            <div class="split_small"></div>
 +
            <div class="fm_22">
 +
                These are the two genetic circuits that we ended up designing.
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            </div>
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            <div class="split"></div>
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            <div class="fb_72"><b>Timed promoter</b></div>
 +
            <br/>
 +
            <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
 +
                freeze it with liquid nitrogen, and find the needed promoter <i>PputA</i> and <i>PglcF</i> through transcriptional
 +
                analysis.
 +
            </div>
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/6/61/T--JiangnanU_China--project_design_1.png"
 +
                style="width: 100%;height: auto;">
 +
            <div class="split_small"></div>
 +
            <div class="fm_22">
 +
                The <i>PputA</i> gene was expressed 5 minutes after phage infection, while the <i>PglcF</i> gene was not expressed
 +
                until 20 minutes after infection (no expression at 5 minutes).
 +
            </div>
 +
            <div class="split_small"></div>
 +
            <img src="https://static.igem.org/mediawiki/2019/a/a5/T--JiangnanU_China--project_design_2.png"
 +
                style="width: 100%;height: auto;">
 +
            <div class="split_small"></div>
 +
            <div class="fm_22">
 +
                We used the fluorescent protein gene and the found promoter to construct two plasmids to introduce into
 +
                <i>E∙coli</i>, and used phage infection to verify whether the promoter was what we wanted.
 +
            </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>
 +
    </div>
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</div>
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</body>
 
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Revision as of 14:34, 14 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.