Difference between revisions of "Part:BBa K5459002"
 
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        <p><strong>Copper Detection Reporter Gene</strong><br>This is a composite part composed of a copper-sensitive promoter BBa_K190017 and a fluorescent protein BBa_K864100. This composite part can function independently or be used in conjunction with BBa_K5459001.<br>In previous iGEM competition projects, such copper ion-sensitive promoters are usually used together with their transcriptional regulatory proteins to detect the concentration of copper ions.<br> This year, we have verified a new possibility: even without introducing additional transcriptional regulatory protein-encoding genes, using only the transcriptional regulatory proteins at the genomic level of E. coli, this copper ion-sensitive promoter can effectively respond to changes in copper ions, fulfill its function, and we have tested the differences between these two schemes.</p>
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            <img src="https://static.igem.wiki/teams/5459/part-registry1/sz-transcription1.png" width="300" " alt="图一" style="width: 300px; margin-right: 10px;">
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<br>In the future, the transformation of other E. coli endogenous regulatory systems can also consider this scheme.<br><strong>PartI：Dual Plasmid System Sensing Copper Ions (Used in Conjunction with BBa_K5459001)</strong></p>
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            <img src="https://static.igem.wiki/teams/5459/heading-imgs/part-registry/cu-with-outer-protein.jpg" width="300" " alt="图一" style="width: 300px; margin-right: 10px;">
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            <img src="https://static.igem.wiki/teams/5459/heading-imgs/part-registry/hill-function-with-outer-protein.jpg" width="200" " alt="图二" style="width: 200px;">
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        <p><strong>Part II: Single Plasmid Sensing Copper Ions</strong></p>
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            <img src="https://static.igem.wiki/teams/5459/heading-imgs/part-registry/cu-without-outer-protein.jpg" alt="图三" style="width: 300px; margin-right: 10px;">
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            <img src="https://static.igem.wiki/teams/5459/heading-imgs/part-registry/hii-function-without-outer-protein.jpg" alt="图四" style="width: 200px;">
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      <p><strong>PartIII :Comparison between Single Plasmid System and Dual Plasmid System</strong></p>
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            <img src="https://static.igem.wiki/teams/5459/heading-imgs/part-registry/compration.jpg" alt="图三" style="width: 200px; margin-right: 10px;">
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        <p>Through the experiment, we concluded that our designed single-plasmid system can also generate a fluorescent signal in a copper-rich environment. This system utilizes the endogenous regulatory protein of E.coli, meaning the expression level of E.coli,s own regulatory protein is sufficient to activate the additional reporter system. In future designs of regulatory mechanisms, if the regulatory protein is naturally present in E.coli,, there may be no need to encode this protein on a plasmid for exogenous expression. Instead, we can attempt to directly use the regulatory protein expressed from the genome, thereby reducing the burden on the engineered bacteria.<br>Upon comparison, we found that the results of the dual-plasmid system were not as strong as those of the single-plasmid system in terms of signal strength. However, the overall curve became steeper, and the induction threshold for low copper ion concentrations was lower. This leads us to speculate that different expression levels of CueR might cause different system responses.</p>
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<p><strong>Reference</strong><br>
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[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>
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[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>
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[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>
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[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>
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[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>
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[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>
 +
[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.<br>
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5459002 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K5459002 parameters</partinfo>
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Latest revision as of 06:46, 2 October 2024

Copper Detection Reporter Gene
This is a composite part composed of a copper-sensitive promoter BBa_K190017 and a fluorescent protein BBa_K864100. This composite part can function independently or be used in conjunction with BBa_K5459001.
In previous iGEM competition projects, such copper ion-sensitive promoters are usually used together with their transcriptional regulatory proteins to detect the concentration of copper ions.
This year, we have verified a new possibility: even without introducing additional transcriptional regulatory protein-encoding genes, using only the transcriptional regulatory proteins at the genomic level of E. coli, this copper ion-sensitive promoter can effectively respond to changes in copper ions, fulfill its function, and we have tested the differences between these two schemes.

图一 图二

In the future, the transformation of other E. coli endogenous regulatory systems can also consider this scheme.
PartI：Dual Plasmid System Sensing Copper Ions (Used in Conjunction with BBa_K5459001)

图一 图二

Part II: Single Plasmid Sensing Copper Ions

图三 图四

PartIII :Comparison between Single Plasmid System and Dual Plasmid System

图三

Through the experiment, we concluded that our designed single-plasmid system can also generate a fluorescent signal in a copper-rich environment. This system utilizes the endogenous regulatory protein of E.coli, meaning the expression level of E.coli,s own regulatory protein is sufficient to activate the additional reporter system. In future designs of regulatory mechanisms, if the regulatory protein is naturally present in E.coli,, there may be no need to encode this protein on a plasmid for exogenous expression. Instead, we can attempt to directly use the regulatory protein expressed from the genome, thereby reducing the burden on the engineered bacteria.
Upon comparison, we found that the results of the dual-plasmid system were not as strong as those of the single-plasmid system in terms of signal strength. However, the overall curve became steeper, and the induction threshold for low copper ion concentrations was lower. This leads us to speculate that different expression levels of CueR might cause different system responses.

Reference
[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.
[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.
[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.
[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.
[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.
[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.
[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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]