Composite

Part:BBa_K4757999:Design

Designed by: Marik M�ller   Group: iGEM23_Heidelberg   (2023-10-07)


Synthetic expression cassette regulated by terepthalic acid and alkanes for PET and PE sensing


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4202
    Illegal EcoRI site found at 4298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4202
    Illegal EcoRI site found at 4298
    Illegal NotI site found at 1625
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4202
    Illegal EcoRI site found at 4298
    Illegal BglII site found at 212
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4202
    Illegal EcoRI site found at 4298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4202
    Illegal EcoRI site found at 4298
    Illegal NgoMIV site found at 934
    Illegal NgoMIV site found at 2716
    Illegal NgoMIV site found at 3187
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Altough both the insert and vector after PCR amplification was sequence verified (oxford nanopore sequencing), the final assembly and transformation has not been succesfully done, as of now.
No data in-vivo fo the assembled part could be generated. Nontheless the we think the thorough characterization of all the parts, still adds valuble new information to the iGEM registry.

Source

XylS/Pm was stems from the TOL plasmid in Pseudomonas putida (obtained through the pSEVA438);
The sRNA scaffold (SgrS) is natively found in Escherichia coli as part from a sugar transport regulatory network;
AlkS/pAlkB is natively found in Pseudomonas putida (sequence ordered through gene synthesis).
mKate2 originiated from Entacmaea quadricolor (sequence ordered through gene synthesis).

References

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Chen, D., Xu, S., Li, S., Tao, S., Li, L., Chen, S., & Wu, L. (2023). Directly Evolved AlkS-Based Biosensor Platform for Monitoring and High-Throughput Screening of Alkane Production. ACS synthetic biology, 12(3), 832-841. <a href="https://doi.org/10.1021/acssynbio.2c00620">https://doi.org/10.1021/acssynbio.2c00620</a>

Gallegos, M. T., Marqués, S., & Ramos, J. L. (1996). Expression of the tol plasmid xylS gene in pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and Alpha 54-dependent tandem promoters according to the compound used for Growth. Journal of Bacteriology, 178(8), 2356-2361. https://doi.org/10.1128/jb.178.8.2356-2361.1996

Gawin, A., Valla, S., & Brautaset, T. (2017). The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering. Microbial biotechnology, 10(4), 702-718. <a href="https://doi.org/10.1111/1751-7915.12701">https://doi.org/10.1111/1751-7915.12701</a>

<a>Geyer, R., Jambeck, J. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made.</a> Science advances, 3(7), e1700782. <a href="https://doi.org/10.1126/sciadv.1700782">https://doi.org/10.1126/sciadv.1700782</a>

Gottesman S. (2004). The small RNA regulators of Escherichia coli: roles and mechanisms*. Annual review of microbiology, 58, 303-328.</a> <a href="https://doi.org/10.1146/annurev.micro.58.030603.123841">https://doi.org/10.1146/annurev.micro.58.030603.123841</a>

Kelly, C. L., Harris, A. W. K., Steel, H., Hancock, E. J., Heap, J. T., & Papachristodoulou, A. (2018). Synthetic negative feedback circuits using engineered small RNAs. Nucleic acids research, 46(18), 9875-9889. <a href="https://doi.org/10.1093/nar/gky828">https://doi.org/10.1093/nar/gky828</a>

Li, J., Nina, M. R. H., Zhang, X., & Bai, Y. (2022). Engineering Transcription Factor XylS for Sensing Phthalic Acid and Terephthalic Acid: An Application for Enzyme Evolution. ACS synthetic biology, 11(3), 1106-1113. <a href="https://doi.org/10.1021/acssynbio.1c00275">https://doi.org/10.1021/acssynbio.1c00275</a>

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Modi, S. R., Camacho, D. M., Kohanski, M. A., Walker, G. C., & Collins, J. J. (2011). Functional characterization of bacterial sRNAs using a network biology approach. Proceedings of the National Academy of Sciences of the United States of America, 108(37), 15522-15527. <a href="https://doi.org/10.1073/pnas.1104318108">https://doi.org/10.1073/pnas.1104318108</a>

Møller, T., Franch, T., Højrup, P., Keene, D. R., Bächinger, H. P., Brennan, R. G., & Valentin-Hansen, P. (2002). Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Molecular cell, 9(1), 23-30. <a href="https://doi.org/10.1016/s1097-2765(01)00436-1">https://doi.org/10.1016/s1097-2765(01)00436-1</a>

Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature biotechnology, 31(2), 170-174. <a href="https://doi.org/10.1038/nbt.2461">https://doi.org/10.1038/nbt.2461</a>

Sharma, S.R. (2018). Bioremediation of Polythenes and Plastics: A Microbial Approach. In: Prasad, R., Aranda, E. (eds) Approaches in Bioremediation. Nanotechnology in the Life Sciences. Springer, Cham. <a href="https://doi.org/10.1007/978-3-030-02369-0_6">https://doi.org/10.1007/978-3-030-02369-0_6</a>

Storz, G., Vogel, J., & Wassarman, K. M. (2011). Regulation by small RNAs in bacteria: expanding frontiers. Molecular cell, 43(6), 880-891. <a href="https://doi.org/10.1016/j.molcel.2011.08.022">https://doi.org/10.1016/j.molcel.2011.08.022</a>

Tournier, V., Topham, C. M., Gilles, A., David, B., Folgoas, C., Moya-Leclair, E., Kamionka, E., Desrousseaux, M. L., Texier, H., Gavalda, S., Cot, M., Guémard, E., Dalibey, M., Nomme, J., Cioci, G., Barbe, S., Chateau, M., André, I., Duquesne, S., & Marty, A. (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219. <a href="https://doi.org/10.1038/s41586-020-2149-4">https://doi.org/10.1038/s41586-020-2149-4</a>

Wu, P., Wang, Z., Zhu, Q., Xie, Z., Mei, Y., Liang, Y., & Chen, Z. (2021). Stress preadaptation and overexpression of rpoS and hfq genes increase stress resistance of Pseudomonas fluorescens ATCC13525. Microbiological research, 250, 126804. <a href="https://doi.org/10.1016/j.micres.2021.126804">https://doi.org/10.1016/j.micres.2021.126804</a>

Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. <a href="https://doi.org/10.1038/srep10907">https://doi.org/10.1038/srep10907</a>