Part:BBa_J45995

Stationary phase dependent GFP generator

BBa_J45995 is a composite part consisting of an Escherichia coli osmY stationary phase promoter (BBa_J45992) and a GFP generator (BBa_E0840). Thus, BBa_J45995 produces fluorescence in stationary phase cultures.

Usage and Biology

See BBa_J45992 for details on the osmY stationary phase promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 872
  

William and Mary iGEM 2022

The 2022 William and Mary iGEM team created two composite parts to improve part BBa_J45995. One of our parts is BBa_K4174001 and the other is BBa_K417002. Like BBa_J45995, these parts use an osmY promoter, which is induced by the host 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). In part BBa_K4174001, this promoter is partnered with a mRFP1 coding region, and this construct fluoresces red once the host cell has entered stationary phase. In part BBa_K417002, this promoter is partnered with a sfGFP coding region, and this construct fluoresces green once the host cell has entered stationary phase. More information about the design and characterization of each of these parts can be found below.

BBa_K417001 Design & Characterization
• In this composite part, we have replaced GFP with mRFP1 from Mutalik et al. 2013 and added unique nuclotide sequences (UNSs) 1 and 10 sequences (Torella et al, 2014) to the ends of the construct.
• 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 sequences to make this part compatible with Gibson Assembly with our pSB1C3 backbone, as we also added UNS1 and UNS10 to our backbone.

To test the effectiveness of our mRFP1 construct (BBa_K4174001), our team transformed the original MIT iGEM 2006 construct (BBa_J45995), our mRFP1 construct, and our sfGFP construct (BBa_K4174001) into E. coli NEB5α cells and grew the various transformants in a plate reader. They were grown at 37°C. 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.

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.

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.

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 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.

BBa_K417002 Design & Characterization

In this composite part, 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 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.

To test the effectiveness of our sfGFP construct (BBa_K4174002), our team transformed the original MIT iGEM 2006 construct, our improved sfGFP construct, and our improved RFP construct (BBa_K4174001) in E. coli NEB5α in a plate reader. The various transformants were grown at 37°C. 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.

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 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.

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 construct. Here, sfGFP is on the far right, and is visibly more brightly green that the original GFP construct.

Sources

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

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

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.

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

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