Part:BBa_K4174002:Design
osmY-sfGFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 419
- 1000COMPATIBLE WITH RFC[1000]
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 green 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 the sfGFP sequence from Ceroni et al. 2015, replaced RBS BBa_B0030 with an RBS containing region from Ceroni et al. 2015, removed the scar sequences, and added unique nucleotide sequences (UNSs) 1 and 10 (Torella et al., 2014) to the ends of the construct.
- We elected to use super-folder green fluorescent protein (sfGFP) as opposed to the original GFP, as it folds more readily in Escherichia coli, thus allowing for a more effective assay (Pédelacq 2006). This sfGFP sequence is codon-optimized for E. coli and was designed by Ceroni et al. (2015) using DNA2.0 for high levels of expression in E. coli (Ceroni et al. 2015).
- We switched the original RBS with an RBS containing region used with the sfGFP sequence in the paper by Ceroni et al. (2015). The RBS containing region by Ceroni et al. was designed using the RBS Calculator (Arpino et al. 2013), and was used by Ceroni et al. with their sfGFP sequence, so we elected to use this RBS and coding region together.
- We added UNS1 and UNS10 sequences to make this part compatible with Gibson Assembly with our backbone, as we also added UNS1 and UNS10 to our pSB1C3 backbone.
- We also designed a similar red fluorescence system. To see information about this, visit parts page BBa_K4174001.
Source
Ceroni, F., Algar, R., Stan, G., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature Methods, 12(5):415-418. Doi: 10.1038/nmeth.3339
References
Arpino, J., Hancock, E. J., Anderson, J., Barahona, M., Stan, G. V., Papachristodoulou, A., & Polizzi, K. (2013). Tuning the dials of Synthetic Biology. Microbiology (Reading, England), 159(Pt 7), 1236–1253. doi.org/10.1099/mic.0.067975-0
Ceroni, F., Algar, R., Stan, G., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature Methods, 12(5):415-418. Doi: 10.1038/nmeth.3339
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.
Pédelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature biotechnology, 24(1), 79-88.