Composite

Part:BBa_K4174001:Design

Designed by: Beteel Abu-Ageel   Group: iGEM22_William_and_Mary   (2022-09-30)
Revision as of 20:00, 10 October 2022 by Bnabuageel (Talk | contribs) (→‎Design Notes)

Design Notes

This part uses an osmY promoter since this promoter is induced by the cell's entry into stationary phase. In typical E. coli cells, osmY, which helps cells transition into stationary phase when under osmotic or metabolic stress, is not produced during exponential growth phase but is produced during stationary phase. Specifically, the osmY promoter is induced by the rpoS (ribosome polymerase sigma S) at the onset of stationary phase (Chang 2002). Because this promoter is partnered with a mRFP1 coding region, this construct fluoresces red once the cell has entered stationary phase.

  • This composite part is an improvement of the 2006 MIT iGEM team's composite part BBa_J45995, which is a stationary phase detector utilizing osmY. We have replaced GFP with mRFP1 from Mutalik et al. 2013 and added UNS1 and UNS10 sequences to the ends.
  • We elected to use monomeric red fluorescent protein (mRFP1) as opposed to the original GFP in order to provide an alternative reporter to GFP for the detection of stationary phase in bacterial cells. mRFP1 is a monomer, has a rapid maturity rate, and has minimal spectral overlap with GFP compared to the wild-type red fluorescent protein DsRed (Campbell et al. 2002). Creating alternative versions of fluorescent bioreporters using proteins with different excitation and emission wavelengths allows researchers to assay multiple parameters at a time, as different fluorescent assays conducted simultaneously must use proteins with different absorption spectra in order for researchers to differentiate between them.
  • The sequence of our mRPF1 construct is compatible with the Type IIS assembly method, unlike the original construct. Constructs uploaded to the iGEM Parts Registry must be compatible with either Type IIS assembly or BioBrick RFC[10] assembly. The original MIT iGEM 2006 construct was only compatible with the BioBrick RFC[10] assembly method, but after altering the sequence as described above, our composite part is now compatible with both Type IIS assembly and BioBrick RFC[10] assembly.
  • We added UNS1 and UNS10 sequences to make this part compatible with Gibson Assembly with the pSB1C3 backbone, as we also added UNS1 and UNS10 to this backbone.

We also designed a similar green fluorescence system using sfGFP. For more information about this, visit parts page BBa_K4174002.

Source

Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q. A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., & Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature methods, 10(4), 354–360. doi.org/10.1038/nmeth.2404

References

Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., & Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 99(12), 7877–7882. doi.org/10.1073/pnas.082243699

Chang, D. E., Smalley, D. J., & Conway, T. (2002). Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model. Molecular microbiology, 45(2), 289-306.

Jaishankar, J., & Srivastava, P. (2017). Molecular basis of stationary phase survival and applications. Frontiers in microbiology, 8, 2000.

Mutalik, V. K., Guimaraes, J. C., Cambray, G., Lam, C., Christoffersen, M. J., Mai, Q. A., Tran, A. B., Paull, M., Keasling, J. D., Arkin, A. P., & Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature methods, 10(4), 354–360. doi.org/10.1038/nmeth.2404