Part:BBa_K5460000
Reporting assay with Standardized Interfaces
This part serves as a reporting module (R module) of our system. In some cases, this plasmid needs to be used in conjunction with our A module. To align with our hardware system for simultaneous multi-biomarker detection, we require various biomarker-sensitive promoters and different fluorescent proteins to avoid signal cross-talk.
We optimized and improved the plasmid system by introducing a GoldenGate interface at key sites within this module. Specifically, we designed a standardized GoldenGate interface at the positions of the promoter and the fluorescent protein.
This allows us to easily incorporate different promoters into this plasmid system through a simple Golden Gate reaction. Different fluorescent signal channels report different biomarkers, providing a biotechnological foundation for our hardware system. Using this reporting system, we tested three different fluorescent proteins—mKate2, sfGFP, and mTagBF2—to report on four different biomarkers: uric acid, glucose, tryptophan, and lactic acid.
Through experimental testing, we successfully obtained the signal intensities corresponding to different concentrations of these biomarkers, validating the feasibility of our system.
Subsequently, we generated response curves from these data and performed Hill equation fitting:
In the future, this system can be adapted to detect other biomarkers by replacing the plasmid accordingly. Additionally, we have developed a hardware system designed to work in conjunction with this detection system. For more details, please refer to our Hardware page.
Reference
[1] https://2019.igem.org/Team:QHFZ-China/Description
[2] https://2019.igem.org/Team:Hong_Kong_LFC_PC/Design
[3] https://2017.igem.org/Team:Hong_Kong_UCCKE
[4] https://2018.igem.org/Team:DLUT_China/Description
[5] https://2022.igem.wiki/sesame-shenzhen/proof-of-concept
[6] Geraths, C., Christen, E.H. & Weber, W., 2012. A hydrogel sensing pathological urate concentrations. Macromolecular Rapid Communications, 33(24), pp.2103-2108. doi:10.1002/marc.201200563.
[7] Subach, O.M., Cranfill, P.J., Davidson, M.W. & Verkhusha, V.V., 2011. An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore. PLoS One, 6(12), p.e28674. doi:10.1371/journal.pone.0028674.
[8] Yan, Q. & Fong, S.S., 2017. Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli. Journal of Biological Engineering, 11, p.33. doi:10.1186/s13036-017-0075-2.
[9] Papakostas, K. & Frillingos, S., 2012. Substrate selectivity of YgfU, a uric acid transporter from Escherichia coli. Journal of Biological Chemistry, 287(19), pp.15684-15695. doi:10.1074/jbc.M112.355818.
[10] Zhang, L.Y., Lin, R.T., Chen, H.R., et al., 2021. High glucose activated cardiac fibroblasts by a disruption of mitochondria-associated membranes [retracted in: Frontiers in Physiology, 2022, 13, p.1006459]. Frontiers in Physiology, 12, p.724470. Published on 18 August 2021.
[11] Shemiakina, I., Ermakova, G., Cranfill, P. et al., 2012. A monomeric red fluorescent protein with low cytotoxicity. Nature Communications, 3, p.1204. doi:10.1038/ncomms2208.
[12] Chevalet, L., Robert, A., Gueneau, F., Bonnefoy, J.Y. & Nguyen, T., 2000. Recombinant protein production driven by the tryptophan promoter is tightly controlled in ICONE 200, a new genetically engineered E. coli mutant. Biotechnology and Bioengineering, 69(4), pp.351-358.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 346
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 346
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 346
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 346
- 1000COMPATIBLE WITH RFC[1000]
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