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| Hydroponics | | Hydroponics |
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− | Naringenin Operon
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− | Improved Operon
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− | Primer construction
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− | <!-- services
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− | <section id='construct' class="s-services">
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− | <div class="row section-header has-bottom-sep" data-aos="fade-up">
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− | <h3 class="subhead">Naringenin Operon</h3>
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− | <h1 class="display-1">DESIGN</h1>
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− | <div class="row about-desc" data-aos="fade-up">
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− | <p class="about-para"> In order to produce the flavonoid naringenin, construction of a naringenin operon that would be synthesised from the sequence in the iGEM parts registry to gblocks by IDT was required. This operon was based on that used by the TU Darmstadt 2014 iGEM team, allowing the production of naringenin to attract nitrogen fixing bacteria. It contains the genes for four enzymes: 4 – Coumaryl ligase, Tyrosine ammonia lyase, Chalcone isomerase and Chalcone synthase. Each of these genes will contain a strong ribosome binding site. This construct will be under the control of a J23100 constitutive promoter to observe its expression in <i>Escherichia coli</i> as a proof of concept. Once biosynthesis under the control of J23100 is achieved, the construct future experiments would test this under a T7 promoter in E. coli. Parts for biosynthesis in the final chassis organism, root-colonising <i>Pseudomonas fluorescens</i>, will be constructed in the plasmid backbone pSB1C3.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/e/e8/T--Newcastle--j23100plasmid.jpeg">
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− | <p style="text-align:center"><br> The naringenin biosynthetic operon under control of a J23100 promoter created in Benchling.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/d/d6/T--Newcastle--t7plasmid.jpeg">
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− | <p style="text-align:center"><br>The naringenin biosynthetic operon under control of a T7 promoter created in Benchling.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/9/95/T--Newcastle--SBOLJ23100.jpeg">
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− | <p style="text-align:center"><br> The naringenin biosynthetic operon construct under control of a J23100 promoter created in SBOL.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/3/31/T--Newcastle--SBOLT7.jpeg">
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− | <p style="text-align:center"><br>The naringenin biosynthetic operon contruct under control of a T7 promoter created in SBOL.</p>
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− | <!-- about
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− | <section id='operon' class="s-about">
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− | <h3 class="subhead subhead--dark">Improved Naringenin Operon based on Pathway Modelling</h3>
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− | <h1 class="display-1 display-1--light">DESIGN</h1>
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− | <p class="about-para"> Results of the modelling showed that weaker expression of the first two genes and 10 fold stronger expression of the last two genes would reduce the build-up of malonyl CoA and optimise naringenin synthesis. Therefore we found two synthetic promoters: BG28 and BG51 and an additional <i>E. coli</i> his operon terminator to be placed after the first two genes. These will allow enhanced naringenin production in future experiments, as BG28 is a weak promoter for the first two genes and BG51 is a strong promoter for the last two.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/e/ed/T--Newcastle--BGplasmid.jpeg">
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− | <p style="text-align:center"><br>The naringenin biosynthetic operon under control of synthetic promoters BG28 and BG51 created in Benchling.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/c/c8/T--Newcastle--BGcloseup.jpeg">
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− | <p style="text-align:center"><br>A close up of the synthetic promoters BG28 and BG51 placement in the operon, created in Benchling.</p>
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− | <img src="https://static.igem.org/mediawiki/2018/c/c5/T--Newcastle--SBOLBG28BG51.jpeg">
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− | <p style="text-align:center"><br> The naringenin biosynthetic operon contruct under control of a BG28 and BG51 promoters and an additional his operon terminator created in SBOL.</p>
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− | <!-- </div> end about-list -->
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