Part:BBa_J45995:Experience
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how you used this part and how it worked out.
Applications of BBa_J45995
Stationary phase dependent fluorescence.
User Reviews
UNIQ3a3d1307ab31a321-partinfo-00000000-QINU
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BBa_J45995 produced fluorescence only in stationary phase. |
UNIQ3a3d1307ab31a321-partinfo-00000003-QINU
Characterization
Transcriptional control of GFP generator
[Note: BBa_J45995 is a composite part of BBa_J45992 and BBa_E0840.]
We successfully designed, constructed and tested transcriptional control devices for constitutive, stationary phase dependent and exponential phase dependent protein production (A-C). To test and verify function of our three transcriptional control devices, we assembled each control device with the GFP protein generator BBa_E0840 and monitored the fluorescence of E. coli cultures with each device over time. For each device, we plot the change in fluorescence per unit time (normalized GFP synthesis rate) versus the cell density (OD600nm) (D). The constitutive transcriptional control device produced a high GFP synthesis rate irrespective of cell density. The stationary phase transcriptional control device produced a low initial GFP synthesis rate which increased with culture cell density. The exponential phase transcriptional control device produced an initially high GFP synthesis rate which dropped off as cell density increased. Data shown are averages of triplicate measurements of cultures grown from three individual colonies of each device. Error bars are the standard deviation of the three individual cultures.
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 of these parts can be found on their respective iGEM Registry pages.
BBa_K4174001
To test the effectiveness of our mRFP1 construct, our team grew the original construct (BBa_J45995), our improved mRFP1 construct (BBa_K4174001), and our improved sfGFP construct (BBa_K4174002) in E. coli NEB5α in a plate reader. They were grown at 37°C using 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.
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.
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 than the original GFP construct.
BBa_K4174002
To test the effectiveness of our sfGFP construct, our team grew the original MIT 2006 construct, our improved sfGFP construct (BBa_K4174002), and our improved mRFP1 construct (BBa_K4174001) 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. The values for green fluorescence are reported below.
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 green than 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. https://doi.org/10.1038/nmeth.2404
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.