Difference between revisions of "Part:BBa K4174001"

Revision as of 22:29, 8 October 2022

osmY-mRFP1

This composite part is composed of osmY promoter, the B0030 RBS, mRFP1, and the B0015 terminator.

Usage and Biology

Sequence and Features

Biological Relevance

The ability to assay whether a chassis is actively transcribing its circuit to make proteins is crucial for testing the efficacy of a fieldable construct. Bacteria have two main life stages - exponential growth, during which they reproduce and express their circuits with ease, and stationary phase, during which they cease most non-essential metabolic activity. Since stationary phase is induced by inopportune environments, such as metabolic shortage, most bacteria in nature exist in stationary phase (Jaishankar 2000). This is a major roadblock for fieldable synthetic biology, as constructs that work perfectly in the lab may stop expressing their circuit when introduced into their deployment sites. In order to assay how a circuit will behave in nature, constructs should be tested in the lab while in stationary phase.

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 sfGFP 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 unique nucleotide sequences (UNS) 1 and 10 sequences (Torella et al., 2014) to the ends.
• We elected to use monomeric red fluorescent protein (mRFP1) as opposed to the original GFP, as it allows for the construction of an alternative fluorescent reporter. 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). However, other DsRed variants have a much higher fluorescence quantum yield and extinction coefficient than mRFP1 (Campbell et al. 2002).
• We added UNS1 and UNS10 (Torella et al., 2014) to make this part compatible with Gibson Assembly with our pSB1C3 backbone, as we also added UNS1 and UNS10 to our backbone.
• We also designed a similar green fluorescence system using sfGFP. For more information about this, visit parts page BBa_K4174002.

Testing and Results

To test the effectiveness of our improved parts, our team grew the original construct (BBa_J45995), our mRFP1 construct, and our sfGFP construct (BBa_K4174001) in E. coli NEB5α in a plate reader. They were grown at 37°C using continuous shaking. For red fluorescence measurements, we used an excitation value of 584 nm and an emission value of 610 nm. For green fluorescence measurements, we used an excitation value of 485 nm and an emission value of 528 nm. The values for red fluorescence are reported below. For information about green fluorescence measurements, see parts page BBa_K4174002.

Based on the graph above, the bacterial cells engineered with our mRFP1 construct appear to have entered stationary phase around 16 hours. As seen in the graph, our mRFP1 construct produces more red fluorescence than the original circuit. The other measurements taken are for our sfGFP construct and untransformed E. coli NEB5α cells, both of which serve as negative controls for red fluorescence.

As seen in the image above, qualitative results reveal that our mRFP1 construct produces more red fluorescence than the original construct. Here, our mRFP1 construct is on the far left, and is visibly more red that the original GFP construct.

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. https://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. https://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.

Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2014). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, 42(1), 681–689. https://doi.org/10.1093/nar/gkt860