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| − | </head>
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| − | <body>
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| − | <div class="bgd" id="head">
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| − | <div class="split_small"></div>
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| − | <div class="split"></div>
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| − | <div class="contents" style="color: white">
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| − | <div class="column">
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| − | <div class="centers">
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| − | <div class="fb_72">
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| − | <b>Results</b>
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| − | </div>
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| − | <br/>
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| − | <div class="fm_22">
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| − | In order to make <i>E. coli</i> in the laboratory resistant to phage infection, this year our team
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| − | first
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| − | found components that responded to phage infection through transcriptome analysis.
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| − | Then we found components that could make the bacteria resistant to phage infection through
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| − | literature search and mutagenesis screening.
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| − | </div>
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| − | <div class="split"></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:auto;">
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| − | <div class="fb_48" style="margin-left: 2%;margin-top: 1%">View all</div>
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| − | </div>
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| − | </a>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − |
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| − | <div class="split"></div>
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| − | <div class="contents" id="phage">
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| − | <div class="column">
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| − | <div class="centers">
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| − |
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| − | <div class="fb_72">
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| − | Phage Isolation
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <div class="fm_22">
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| − | We added 1 μL of phage-infected fermentation broth to a plate containing <i>E. coli</i> BL21.
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| − | <br/>
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| − | After proper culture for a period of time, plaque appeared on the plate (Fig.1.)
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| − | <br/>
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| − | We isolated the phages from the plate and photographed them using a projective electron microscope (Fig.
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| − | 2).
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| − | <br/>
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| − | We finally determined that the T4 phages infected our fermentation broth by sequencing the genome of the
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| − | phages.
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/2/2a/T--JiangnanU_China--results_2.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − |
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| − | <!--第二部分-->
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| − | <div class="split"></div>
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| − | <div class="fb_72"><b>Selection of Inducible Promoters</b></div>
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| − | <div class="split"></div>
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| − | <div class="fb_48">
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| − | 1.Selection
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <div class="fm_22">
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| − | In order to screen inducible promoters, we first made the one-step growth curve of phages (Fig. 3).
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| − | After that, we selected two time points of phage infection for 5min （in the incubation period of phage
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| − | infection）and phage infection for 20min （in the outbreak period of phage infection）through the one-step
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| − | growth curve of phage.
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| − | By analyzing transcriptome data，we selected inducible promoter P<i>putA</i> (Fig.4) for 5 min and
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| − | inducible
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| − | promoter P<i>glcF</i> (Fig.5) for 20 min.
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/e/e2/T--JiangnanU_China--results_3.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/e/e8/T--JiangnanU_China--results_1.png"
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| − | style="width: 100%;height: auto;">
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| − |
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| − | <!-- 第二部分-->
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| − | <div class="split_small"></div>
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| − | <div class="fb_48">
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| − | 2. Characterization
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <div class="fm_22">
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| − | In <i>E. coli</i> BL21，we connected the green fluorescence gene <i>gfp</i> with the inducible promoter P<i>putA</i>
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| − | for 5 min
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| − | (Fig.6) and the red fluorescence gene <i>mCherry</i> with the inducible promoter P<i>glcF</i> for 20 min
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| − | (Fig.7) in
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| − | our genetic circuits.
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| − | After infecting the bacteria with phages for the corresponding time, we observed that the infected cells
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| − | gave off green and red fluorescence respectively.
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| − |
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/b/be/T--JiangnanU_China--results_0.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <!-- 第三部分-->
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| − | <div class="split_small"></div>
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| − | <div class="fb_48">
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| − | 3. Anti-phage Parts
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <div class="fm_22">
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| − | When we had components that responded to phage infection, we searched for anti-phage parts through
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| − | literature search and mutagenesis screening.
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <div class="fm_22">
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| − | 3.1 Anti-phage part from literature
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| − | <br/>
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| − | Through literature search, we found a resistant protein AbpAB that can resist T4 phage. Protein AbpAB
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| − | can impair the synthesis of the phage DNA and late gene transcripts, which resulted in poor expression
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| − | of late proteins and consequently no phage propagation. AbpAB have no effect on the bacterial growth(Fig
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| − | 8.) which is important to the industry. However, protein AbpAB didn’t work well as we expected(Fig 9.).
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/b/b4/T--JiangnanU_China--result_4.png"
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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">
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| − | 3.2. Anti-phage part from mutation screening
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| − | <br/>
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| − | To get more efficient phage resistant parts, we used the ARTP(Atmospheric and room temperature plasma)
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| − | mutagenesis system to obtain a large number of mutant strains. Then we co-cultured the mutant strains
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| − | with the phage and screened the mutant strains that could resist phage infection. In the screening
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| − | process, we continuously verified their resistance, eliminated the bacterial strains with degraded
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| − | resistance and retained the ones with excellent resistance. Then we obtained 8 mutant strains which were
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| − | resistant to phage and four key mutation sites (nuoE, yhjH, rzpD, and gntR) through comparation of
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| − | genome (Fig. 10). Resistance tests on these key sites were performed respectively(Fig 11.). According to
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| − | the advice of corporate stakeholders, if the Genetic Modified (GM) strain is to be applied in industry,
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| − | our components cannot have a great influence on bacterial growth. Since it is not possible to directly
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| − | see from the figure which component has the least influence on the growth of the bacteria, we use the
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| − | Grey Relation Analysis（GRA） method to analyze the growth curve（Fig 12.） of the bacteria connecting the
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| − | various components. We used the Entropy Weight Method (EWM) to determine the weight of each growth point
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| − | to select the most similarly modified strain (the highest correlation), which is the component that has
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| − | the least impact on bacterial growth. At the same time, after consulting the industry experts, we
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| − | revised the weights according to the experts' recommendations to evaluate the components again, making
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| − | them more in line with the real situation of production, that is, the most suitable components for
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| − | industrial production. Using GRA's two evaluations of the four components at different weights, we
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| − | selected the component gntR.(Fig 13.)
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/7/73/T--JiangnanU_China--result_5.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/3/3c/T--JiangnanU_China--result_6.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/1/16/T--JiangnanU_China--result_7.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/3/31/T--JiangnanU_China--result_8.png"
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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">
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| − | 3.3. Cascade of protein AbpAB and GntR
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| − | <br/>
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| − | First, resistant proteins AbpAB and GntR were verified by SDS-PAGE（Fig 14.）Then，We connected gntR with
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| − | abpAB in pET-28a plasmid , and transformed them into E. coli BL21. We co-expressed abpAB and gntR, and
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| − | subsequently obtained a recombinant strain that is resistant to phage（Fig 15.）. The result showed that
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| − | two proteins works better together.
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/a/a8/T--JiangnanU_China--result_9.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/5/59/T--JiangnanU_China--result_10.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − |
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| − | <!-- 第四部分-->
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| − | <div class="split_small"></div>
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| − | <div class="fb_48">
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| − | 4. Assembly and application
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <div class="fm_22">
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| − | In order to prevent the phage from escaping the attack of our resistant protein, we connected a kill
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| − | switch p-1 (BBa_K628000) with the 20 min induction promoter(Fig 16).
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--JiangnanU_China--result_11.png"
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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">
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| − | We inoculated E. coli BL21 and recombinant E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 in LB liquid
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| − | medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Then the fresh phage solution was
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| − | inoculated at the same time and continuous cultured for 1-2 h. As a result, it was found that the
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| − | recombinant grew well.(Fig 17.).
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/d/d5/T--JiangnanU_China--result_12.png"
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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">
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| − | In addition, we cooperated with Ningxia EPPEN BIOTECH CO.,LTD (http://www.eppen.com.cn) to carry out
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| − | small-scale and pilot test fermentation experiments of resistant strain in the special fermentation
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| − | laboratory of Jiangnan University. This ensured that our experiments were controllable without any phage
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| − | and engineered bacteria leaking.
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| − | <br />
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| − | We transferred the constructed plasmid into an engineering bacteria strain producing γ-aminobutyric acid
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| − | (GABA).
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| − | <br />
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| − | The fermentation laboratory is subjected to UV irradiation and ozone fumigation prior to formal
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| − | fermentation to remove phage that may be present. The E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1, E.
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| − | coli BL21-pET28a-PputA-abpAB and the control （E. coli BL21） were added T4 phage after six hours of
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| − | culture, and the fermentation was continued for 10 hours. During the fermentation, the OD was measured,
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| − | and the effects of the phage on the three were observed.
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| − | <br />
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| − | Then we used whole-cell transformation with a combination of resistant strain E. coli
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| − | BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1, and we got a good whole-cell transformation ability of the
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| − | resistant strain (Figure 18).
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| − | <br />
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| − | From the results, our resistant composite part has great advantages in the production of γ-aminobutyric
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| − | acid and are not threatened by T4 phage. The productivity of γ-aminobutyric acid is 278.3 g/L, and the
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| − | molar conversion rate is really high, reaching 98.4%(Table 1.), which means that the circuit we built
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| − | can be used in production without any impact (Figure 19).
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| − | </div>
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/7/7a/T--JiangnanU_China--result_13.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <img src="https://static.igem.org/mediawiki/2019/9/95/T--JiangnanU_China--result_14.png"
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| − | style="width: 100%;height: auto;">
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| − | <div class="split_small"></div>
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| − | <!-- 书签-->
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| − | <div class="split_small"></div>
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| − | <a href="#head"><img src="https://static.igem.org/mediawiki/2019/2/24/T--JiangnanU_China--host_back.png"
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| − | </div>
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| − | </div>
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| − | </html>
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| − | {{:Team:JiangnanU_China/Footer}}
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