Difference between revisions of "Part:BBa K4174001"

Latest revision as of 02:18, 12 October 2022

osmY-mRFP1

This composite part is composed of BBa_K2680553 (UNS1), BBa_J45992 (osmY promoter) followed by a scar sequence (tactagag), the BBa_B0030 RBS followed by a scar sequence (tactag), BBa_K4174003 (mRFP1) followed by a scar sequence (tactagag), the BBa_B0015 terminator, and UNS 10 (BBa_K2680554).

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 an mRFP1 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 in order to provide an alternative reporter to GFP for the detection of stationary phase in bacterial cells. 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). Creating alternative versions of fluorescent bioreporters using proteins with different excitation and emission wavelengths allows researchers to assay multiple parameters at a time, as different fluorescent assays conducted simultaneously must use proteins with different absorption spectra in order for researchers to differentiate between them.
• The sequence of our mRPF1 construct is compatible with the Type IIS assembly method, unlike the original construct. Constructs uploaded to the iGEM Parts Registry must be compatible with either Type IIS assembly or BioBrick RFC[10] assembly. The original MIT iGEM 2006 construct was only compatible with the BioBrick RFC[10] assembly method, but after altering the sequence as described above, our composite part is now compatible with both Type IIS assembly and BioBrick RFC[10] assembly.
• We added UNS1 and UNS10 (Torella et al., 2014) to make this part compatible with Gibson Assembly using the pSB1C3 backbone, as we also added UNS1 and UNS10 to this 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 osmY-mRFP1 construct (BBa_K4174001), our team transformed the original MIT iGEM 2006 osmY construct (BBa_J45995), our osmY-mRFP1 construct, and our osmY-sfGFP construct (BBa_K4174002) into E. coli NEB5-α cells and grew the various transformants in a plate reader. They were grown at 37°C with continuous shaking. For red fluorescence measurements, we used an excitation value of 584 nm and an emission value of 610 nm. The values for red fluorescence are reported below. For information about green fluorescence measurements, see parts page BBa_K4174002.

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 osmY-mRFP1 construct (BBa_K4174001) appear to have entered stationary phase around 16 hours. As seen in the graph, our osmY-mRFP1 construct produces more red fluorescence than the original osmY construct (BBa_J45995). The other measurements taken are for our osmY-sfGFP construct (BBa_K4174002) and untransformed E. coli NEB5-α cells, both of which serve as negative controls for red fluorescence.

The fluorescence intensity of the untransformed cells appears higher than that of cells transformed with our osmY-mRFP1 construct due to our normalization process. Although the raw fluorescence values of the untransformed cells were consistently lower than the raw fluorescence values of the osmY-mRFP1 circuit, the OD600 values of the untransformed cells were much lower than the OD600 values of the cells transformed with osmY-mRFP1. Therefore, when we normalized by dividing raw fluorescence by OD600, the fluorescence intensity of the untransformed cells appeared to be higher than that of the osmY-mRFP1 transformants.

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 osmY-mRFP1 construct (BBa_K4174001) produces more red fluorescence than the original construct. Here, our osmY-mRFP1 construct is on the far left, and is visibly red, unlike our osmY-sfGFP construct (BBa_K4174002) and the original MIT iGEM 2006 osmY construct (BBa_J45995).

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. 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

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.

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