Difference between revisions of "Part:BBa J45995:Experience"

Revision as of 20:39, 10 October 2022

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_J45995

Stationary phase dependent fluorescence.

User Reviews

UNIQ4202f01e591521a1-partinfo-00000000-QINU

UNIQ4202f01e591521a1-partinfo-00000003-QINU

Characterization

Transcriptional control of GFP generator

[Note: BBa_J45995 is a composite part of BBa_J45992 and BBa_E0840.]

Growth phase dependent transcriptional control devices
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 osmY-mRFP1 construct (BBa_K4174001), our team transformed the original MIT iGEM 2006 osmY construct (BBa_J45995), our osmY-mRFP1 construct (BBa_K4174001), 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. For red fluorescence measurements, we used an excitation wavelength of 584 nm and an emission wavelength of 610 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 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 circuit (BBa_J45995). The other measurements taken are for our osmY-sfGFP construct and untransformed E. coli NEB5-α 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 osmY-mRFP1 construct (BBa_K4174001) produces more red fluorescence than the original construct (BBa_J45995). Here, a bacterial culture engineered with our osmY-mRFP1 construct (BBa_K4174001) is on the far left, and is visibly more red than the culture engineered with the original construct (BBa_J45995) and our sfGFP-osmY construct (BBa_K4174002).

BBa_K4174002

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 osmY-sfGFP construct (BBa_K4174002), our team transformed the original MIT iGEM 2006 osmY construct (BBa_J45995), our improved osmY-sfGFP construct (BBa_K4174002), 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.

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 sfGFP circuit is much more fluorescent. The other constructs are our osmY-mRFP1 (BBa_K4174001) 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, a bacterial culture engineered with our osmY-sfGFP construct (BBa_K4174002) is on the far right, and is visibly more green than the culture engineered with the original MIT 2006 iGEM osmY construct (BBa_J45995).

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

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.

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