Difference between revisions of "Part:BBa K4174002"

Revision as of 20:04, 10 October 2022

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

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 (Torella et al., 2014) 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 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 unique nucleotide sequences (UNSs) 1 and 10 (Torella et al., 2014) 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 red fluorescence system. To see information about this, visit parts page BBa_K4174001.

Testing and Results

To test the effectiveness of our osmY-sfGFP construct (BBa_K4174002), our team transformed the original MIT iGEM 2006 osmY construct (BBa_J45995), our improved osmY-sfGFP construct, and our improved osmY-mRFP1 construct (BBa_K4174001) in E. coli NEB5-α in a plate reader. The various transformants were grown at 37°C. For green fluorescence, we used an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The values for green fluorescence are reported below. For information about red fluorescence, see parts page BBa_K4174001.

The data represented in the graph above only includes measurements taken starting from around the 10 hour and 5 minute mark (out of a total growth time of about 19 hours and 35 minutes). In addition, the data shown represents the averages of fluorescence measurements (normalized to OD600) from two experiments.

As seen in the graph above, both the osmY-sfGFP (BBa_K4174002) and MIT iGEM 2006 osmY (BBa_J45995) constructs enter stationary phase right before 14 hours, but our improved osmY-sfGFP construct (BBa_K4174002) is much more fluorescent. The other constructs are our osmY-mRFP1 construct (BBa_K4174001) and untransformed NEB5-α E. coli cells, both of which serve as negative controls for green fluorescence.

Please note that the data in the graph above includes the averages of fluorescence measurements (normalized to OD600) taken from two experiments. For one experiment, we diluted a culture grown overnight in 4 mLs of LB to an OD of 0.1, then loaded the culture into wells to grow overnight. For the other experiment, we inoculated into 1 mL of culture, waited roughly 30 minutes, and loaded the culture into the well plates to grow. For more information on our experimental protocol, please see the Experiments page of the William and Mary iGEM 2022 wiki.

As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original osmY construct (BBa_J45995). Here, a bacterial culture engineered with our osmY-sfGFP construct (BBa_K4174002) is on the far right, and is visibly more green that the culture engineered with the original osmY construct (BBa_J45995).

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.

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. doi.org/10.1093/nar/gkt860