Part:BBa_K4174002
osmY-sfGFP
This is a stationary phase assay utilizing superfolder green fluorescence protein (sfGFP). This composite part is composed of BBa_K2680553 (UNS1), BBa_J45992 (osmY promoter), BBa_K3773008 (RBS containing region), BBa_K3773003 (sfGFP), BBa_B0015 (terminator), and BBa_K2680554 (UNS10).
Sequence and Features
- 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]
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 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 sfGFP 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 UNS1 and UNS10 sequences to the ends.
- 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 serving as a more effective assay (Pédelacq 2006).
- We switched the original RBS with an RBS containing region used with the sfGFP sequence in Ceroni (2015)'s paper. Our team had previously used those parts together successfully, so we elected to use them together again.
- We added unique nucleotide sequences (UNSs) 1 and 10 (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 red fluorescence system. To see information about this, visit parts page BBa_K4174001.
Testing and Results
To test the effectiveness of our improved parts, our team grew the original MIT 2006 construct, our improved sfGFP construct, and our improved RFP construct in E. coli NEB5α in a plate reader. They were grown at 37°C using continuous shaking. For green fluorescence, we used an excitation value of 485 and an emission value of 528. For red fluorescence, we used an excitation value of 584 and an emission value of 610. The values for green fluorescence are reported below. For information about red fluorescence, see parts page BBa_K4174001.
As seen in the graph above, both the sfGFP and MIT GFP constructs enter stationary phase right before 14 hours, but our improved sfGFP circuit is much more fluorescent. The other constructs are our RFP construct and untransformed E. coli cells, both of which serve as negative controls for green fluorescence.
As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original construct. Here, sfGFP is on the far right, and is visibly more brightly green that the original GFP construct.
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
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.
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.
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
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