Difference between revisions of "Team:Shanghai Metro/Contribution"

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    <div class="sub-content">
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        <div class="sub-title">Parts Contribution</div>
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        <div class="article-title">==Contribution==</div>
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        <div class="article-content">BBa_K4028000 is a coding sequence of tke2 from Pseudomonas putida KT2440. Tke2,
 +
            known as a toxic Rhs-type
 +
            effector, is an effect factor in type VI secretion system (T6SS).<br />
 +
            A wide range of Gram-negative bacteria have been shown to have antibacterial T6SSs, including opportunistic
 +
            pathogens such as Pseudomonas aeruginosa,[4] obligate commensal species that inhabit the human gut
 +
            (Bacteroides spp.),[5] and plant-associated bacteria such as Agrobacterium tumefaciens.[6] Under natural
 +
            conditions, bacterial cells encoding T6SS transport effect factors with cytotoxic or antibacterial effects
 +
            (amidase, glycoside hydrolyase, lipase, etc.) to recipient cells through physical contact, thus inhibiting
 +
            the growth of recipient cells. The bacteria encoding T6SS can translate and produce corresponding immune
 +
            proteins to counteract the damage caused by toxic effector factors.[1,2,3]
 +
        </div>
 +
        <div class="img-wrap">
 +
            <img src="https://static.igem.org/mediawiki/2021/7/77/T--Shanghai_Metro--contribution01.png" alt="">
 +
            <span>Figure1. Principle diagram of T6SS.</span>
 +
        </div>
 +
        <div class="article-title">==Reference==</div>
 +
        <div class="article-content">1. Bingle, l.E.H. et al. (2008). Type VI secretion: a beginner’s guide. Current
 +
            opinion in microbiology. 11:3-8.</div>
 +
        <div class="article-content">2. Silverman, J. M. et al. (2012). Structure and regulation of the type VI
 +
            secretion system. 66:453-472.</div>
 +
        <div class="article-content">3. Hernandez, R. E. et al. (2020). Type VI secretion system effector protein:
 +
            Effective weapons for bacterial competitiveness. Cellular microbiology. 22:e13241.</div>
 +
        <div class="article-content">4. Hood RD, . et al (January 2010). "A type VI secretion system ofPseudomonas
 +
            aeruginosa targets a toxin to bacteria". Cell Host & Microbe. 7 (1): 25–37. doi:10.1016/j.chom.2009.12.007.
 +
            PMC 2831478. PMID 20114026.</div>
 +
        <div class="article-content">5. Russell AB, . et al (August 2014). "A type VI secretion-related pathway in
 +
            Bacteroidetes mediates interbacterial antagonism". Cell Host & Microbe. 16 (2): 227–236.
 +
            doi:10.1016/j.chom.2014.07.007. PMC 4136423. PMID 25070807.</div>
 +
        <div class="article-content">6. Ma LS, . et al (July 2014). "Agrobacterium tumefaciens deploys a superfamily of
 +
            type VI secretion DNase effectors as weapons for interbacterial competition in planta". Cell Host & Microbe.
 +
            16 (1): 94–104. doi:10.1016/j.chom.2014.06.002. PMC 4096383. PMID 24981331.</div>
 +
        <div class="article-content">This is the naturally occurring version of the TetR class B promoter. It can be
 +
            induced by tetracycline to express downstream proteins.</div>
 +
        <div class="article-title">==Contribution==</div>
 +
        <div class="article-content">We set different tetracycline concentrations to test the protein expression induced
 +
            by TetR(B) promoter. In our design, the immune protein (ike4) is regulated by TetR(B) promoter. The
 +
            expression of the immune protein (ike4) directly affects and is positively correlated with strain growth.
 +
            Therefore, the strain growth can indicate the regulation of the TetR(B) promoter.</div>
 +
        <div class="article-content">The results of the test are below.</div>
 +
        <div class="img-wrap">
 +
            <span>Table 1. OD<sub>600</sub> value of the strain D: Pus232-ike4-tke4 after 6 hours</span>
 +
            <img src="https://static.igem.org/mediawiki/2021/b/b2/T--Shanghai_Metro--contribution02.png" alt="">
 +
        </div>
 +
        <div class="img-wrap">
 +
            <img src="https://static.igem.org/mediawiki/2021/d/da/T--Shanghai_Metro--contribution03.png" alt="">
 +
            <span>Figure 1. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline</span>
 +
        </div>
 +
        <div class="article-content">The results show that concentrations at the range of 300-500µg/L are more
 +
            effective. The results also proved that adding tetracycline to strain will boost the growth of strain. It
 +
            determines that the optimal concentration of tetracycline is 300µg/L to 500µg/L.</div>
 +
        <div class="article-title">==Contribution==</div>
 +
        <div class="article-content">With this idea coming from the T6SS, we designed to express the effector components
 +
            of T6SS in industrial strains by inserting the gene segment tke2-ike2 to induce the controlled expression of
 +
            the corresponding immune proteins. The normal growth of industrial microorganisms can be ensured only by
 +
            adding certain components (tetracycline) in the fermentation broth, so as to achieve the purpose of the
 +
            experiment. </div>
 +
        <div class="img-wrap">
 +
            <img src="https://static.igem.org/mediawiki/2021/6/67/T--Shanghai_Metro--contribution04.png" alt="">
 +
            <span>Figure1. The gene design of TetR promoter-ike2-lac promoter-tke2.</span>
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            <p class="margin-bottom-8">Email: <i style="color:#0030e4;">binghan423@163.com; runshigu@126.com</i></p>
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<h1>Contribution </h1>
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<H3>
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Bronze Medal Criterion #4
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</H3>
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<p>Make a useful contribution for future iGEM teams. Use this page to document that contribution.
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<br><br>
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Please see the <a href="https://2021.igem.org/Judging/Medals">2021 Medals Page</a> for more information.
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<p>If you are making a contribution by adding information to an existing Part or creating a new Part, you must document your contribution on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria. You can use this page to link to that part and include additional information about your contribution.</p>
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Revision as of 15:31, 14 October 2021

Shanghai_Metro

Parts Contribution
==Contribution==
BBa_K4028000 is a coding sequence of tke2 from Pseudomonas putida KT2440. Tke2, known as a toxic Rhs-type effector, is an effect factor in type VI secretion system (T6SS).
A wide range of Gram-negative bacteria have been shown to have antibacterial T6SSs, including opportunistic pathogens such as Pseudomonas aeruginosa,[4] obligate commensal species that inhabit the human gut (Bacteroides spp.),[5] and plant-associated bacteria such as Agrobacterium tumefaciens.[6] Under natural conditions, bacterial cells encoding T6SS transport effect factors with cytotoxic or antibacterial effects (amidase, glycoside hydrolyase, lipase, etc.) to recipient cells through physical contact, thus inhibiting the growth of recipient cells. The bacteria encoding T6SS can translate and produce corresponding immune proteins to counteract the damage caused by toxic effector factors.[1,2,3]
Figure1. Principle diagram of T6SS.
==Reference==
1. Bingle, l.E.H. et al. (2008). Type VI secretion: a beginner’s guide. Current opinion in microbiology. 11:3-8.
2. Silverman, J. M. et al. (2012). Structure and regulation of the type VI secretion system. 66:453-472.
3. Hernandez, R. E. et al. (2020). Type VI secretion system effector protein: Effective weapons for bacterial competitiveness. Cellular microbiology. 22:e13241.
4. Hood RD, . et al (January 2010). "A type VI secretion system ofPseudomonas aeruginosa targets a toxin to bacteria". Cell Host & Microbe. 7 (1): 25–37. doi:10.1016/j.chom.2009.12.007. PMC 2831478. PMID 20114026.
5. Russell AB, . et al (August 2014). "A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism". Cell Host & Microbe. 16 (2): 227–236. doi:10.1016/j.chom.2014.07.007. PMC 4136423. PMID 25070807.
6. Ma LS, . et al (July 2014). "Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta". Cell Host & Microbe. 16 (1): 94–104. doi:10.1016/j.chom.2014.06.002. PMC 4096383. PMID 24981331.
This is the naturally occurring version of the TetR class B promoter. It can be induced by tetracycline to express downstream proteins.
==Contribution==
We set different tetracycline concentrations to test the protein expression induced by TetR(B) promoter. In our design, the immune protein (ike4) is regulated by TetR(B) promoter. The expression of the immune protein (ike4) directly affects and is positively correlated with strain growth. Therefore, the strain growth can indicate the regulation of the TetR(B) promoter.
The results of the test are below.
Table 1. OD600 value of the strain D: Pus232-ike4-tke4 after 6 hours
Figure 1. Biomass Concentration curve of Pus323-tke4-ike4 Strain with tetracycline
The results show that concentrations at the range of 300-500µg/L are more effective. The results also proved that adding tetracycline to strain will boost the growth of strain. It determines that the optimal concentration of tetracycline is 300µg/L to 500µg/L.
==Contribution==
With this idea coming from the T6SS, we designed to express the effector components of T6SS in industrial strains by inserting the gene segment tke2-ike2 to induce the controlled expression of the corresponding immune proteins. The normal growth of industrial microorganisms can be ensured only by adding certain components (tetracycline) in the fermentation broth, so as to achieve the purpose of the experiment.
Figure1. The gene design of TetR promoter-ike2-lac promoter-tke2.

Contact Info.

Email: binghan423@163.com; runshigu@126.com

Wechat Official Account: IGEM VIPatent