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Commit c0944a03 authored by Jay's avatar Jay
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design fix and engineering fix

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<section class="clean-block clean-post dark"> <section class="clean-block clean-post dark">
<div class="container"> <div class="container">
<div class="block-content"> <div class="block-content">
<div class="post-image" style="background-image:url('assets/img/scenery/image3.jpg');"></div> <div class="post-image" style="background-image:url('https://static.igem.wiki/teams/5459/design.jpg');"></div>
<div class="post-body"> <div class="post-body">
<h2 class="section-heading mb-4"> <h2 class="section-heading mb-4">
<span class="section-heading-lower"><strong>Design</strong></span> <span class="section-heading-lower"><strong>Design</strong></span>
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engineering.html
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important role in regulating copper ion homeostasis and resistance to copper toxicity. When important role in regulating copper ion homeostasis and resistance to copper toxicity. When
the external copper ion concentration increases, CueR binds to copper ions with high the external copper ion concentration increases, CueR binds to copper ions with high
affinity. Once copper ions enter the cell, CueR rapidly binds to them, triggering a affinity. Once copper ions enter the cell, CueR rapidly binds to them, triggering a
conformational change in the CueR protein.</p> conformational change in the CueR protein[1,2].</p>
<p>In the absence of copper ions, CueR binds to the promoter region (pCoA) in a repressive <p>In the absence of copper ions, CueR binds to the promoter region (pCoA) in a repressive
state, preventing the expression of downstream genes. When CueR binds to copper ions, it state, preventing the expression of downstream genes. When CueR binds to copper ions, it
undergoes a conformational change, transitioning from a repressive state to an active state, undergoes a conformational change, transitioning from a repressive state to an active state,
which then initiates the expression of downstream genes. These genes include:</p> which then initiates the expression of downstream genes. These genes include[3]:</p>
<li>CopA: Encodes a copper ion-transporting ATPase that expels excess copper ions from the cell, <li>CopA: Encodes a copper ion-transporting ATPase that expels excess copper ions from the cell,
maintaining copper ion balance. </li> maintaining copper ion balance. </li>
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</ul> </ul>
</li> </li>
<li>2. The second plasmid contains a CueR-sensitive pCoA promoter, with a downstream encoded <li>2. The second plasmid contains a CueR-sensitive pCoA promoter, with a downstream encoded
fluorescent protein: fluorescent protein[4]:
<ul> <ul>
<li>CopA -BBa_B0030- mVenusNB - BBa_B1005</li> <li>CopA -BBa_B0030- mVenusNB - BBa_B1005</li>
</ul> </ul>
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<span id="s11" class="section-heading-upper h4"><br />Design</span> <span id="s11" class="section-heading-upper h4"><br />Design</span>
<p>Copper efflux regulator (CueR) is a Cu+- and Ag+- sensing metalloregulator in E.coli that <p>Copper efflux regulator (CueR) is a Cu+- and Ag+- sensing metalloregulator in E.coli that
controls the expression of two genes involved in metal homeostasis: CopA, which encodes a controls the expression of two genes involved in metal homeostasis: CopA, which encodes a
copper/silver ATPase, and CueO, which encodes a copper oxidase [1]. Like most other copper/silver ATPase, and CueO, which encodes a copper oxidase [5]. Like most other
metalloregulators, CueR acts on promoter DNA that exceeds the optimal length (~17bp) for metalloregulators, CueR acts on promoter DNA that exceeds the optimal length (~17bp) for
recognition by sigma70, a subunit of RNA polymerase (RNAP) [2,3]. Taking advantage of this recognition by sigma70, a subunit of RNA polymerase (RNAP) [5,6]. Taking advantage of this
endogenous Cu(II) sensor system, we designed a simplified prototype plasmid to measure the endogenous Cu(II) sensor system, we designed a simplified prototype plasmid to measure the
concentration of Cu(II) ions. </p> concentration of Cu(II) ions. </p>
<p>This reporting plasmid contains a CueR-regulated copper inducible promoter (BBa_K190017) <p>This reporting plasmid contains a CueR-regulated copper inducible promoter (BBa_K190017)
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<p>We monitored the growth status and fluorescence intensity of the bacterial culture in this <p>We monitored the growth status and fluorescence intensity of the bacterial culture in this
expression system at different copper ion concentrations, as shown in the figure. expression system at different copper ion concentrations, as shown in the figure.
Additionally, after the system reached a steady state, we recorded the final fluorescence Additionally, after the system reached a steady state, we recorded the final fluorescence
values and plotted a response curve against the different copper ion concentrations.</p> values and plotted a response curve against the different copper ion concentrations[7].</p>
<figure class="text-center"> <figure class="text-center">
<div class="image-container d-flex justify-content-between"> <div class="image-container d-flex justify-content-between">
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this model can be consulted on our provided "Model" page. Through this model, we aim to this model can be consulted on our provided "Model" page. Through this model, we aim to
reveal the specific mechanisms by which promoter sequence characteristics affect their reveal the specific mechanisms by which promoter sequence characteristics affect their
initiation activity, providing a theoretical foundation and experimental guidance for initiation activity, providing a theoretical foundation and experimental guidance for
subsequent research on gene expression regulation.</p> subsequent research on gene expression regulation.
</p>
<span id="References" class="section-heading-upper h3"><br />References</span>
<span>
<br />
[1] Hu, Y. & Liu, B., 2024. The copper efflux regulator (CueR). Subcellular Biochemistry, 104, pp.17-31. doi:10.1007/978-3-031-58843-3_2.
<br /><br />
[2] Checa, S.K. & Soncini, F.C., 2011. Bacterial gold sensing and resistance. Biometals, 24(3), pp.419-427. doi:10.1007/s10534-010-9393-2.
<br /><br />
[3] Zhou, X., Xiang, Q., Wu, Y., et al., 2024. A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater. Frontiers in Microbiology, 15, p.1367583. Published on 21 March 2024. doi:10.3389/fmicb.2024.1367583.
<br /><br />
[4] Lischik, C.Q., Adelmann, L. & Wittbrodt, J., 2019. Enhanced in vivo-imaging in medaka by optimized anaesthesia, fluorescent protein selection and removal of pigmentation. PLoS One, 14(3), p.e0212956. Published on 7 March 2019. doi:10.1371/journal.pone.0212956.
<br /><br />
[5] Brown, N.L., Stoyanov, J.V., Kidd, S.P. & Hobman, J.L., 2003. The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2-3), pp.145-163.
<br /><br />
[6] Phillips, C., Canalizo-Hernandez, M., Yidirim, A., Schatz, G.C., Mondragon, A. & O'Halloran, T.V., 2015. Allosteric transcription regulation via changes in the overall topology of the core promoter. Science, 349(6250), pp.877-881.
<br /><br />
[7] Wang, Z.K., Gong, J.S., Su, C., et al., 2024. Multilevel systematic optimization to achieve efficient integrated expression of Escherichia coli. ACS Synthetic Biology, 13(9), pp.2887-2898. doi:10.1021/acssynbio.4c00280.
</span>
</div> </div>
</div> </div>
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