Part:BBa_K5114824
STF: LexA-hER-VP16
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 517
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 517
Illegal NheI site found at 622 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 517
Illegal BamHI site found at 636 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 517
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 517
Illegal AgeI site found at 64 - 1000COMPATIBLE WITH RFC[1000]
The genetic components of the hybrid promoter were taken from previous literature from researchers developing a combinatorial approach to estrogen receptor synthetic transcription factor‐promoter combinations in yeast cells Saccharomyces cerevisiae. The team’s Plex promoter consisted of 3x uvrA sequences and GAL1p 250bp subunits. These sequences were found to have the highest affinity to the STF, leading to the most fluoresence in their experimentations. This PLex promoter binds to Part 5114321 (https://parts.igem.org/Part:BBa_K5114231).
Usage and Biology
PLex Hybrid Promoter: The LexA promoter, an essential native promoter found in E. coli, was engineered with a GAL1 sequence operator, the sequence at which the STF binds to in the promoter region. Taken from the same researchers who developed the estrogen receptor STF, GAL1p 250bp concatenated with 3 units of uvrA, creating the promoter that showed the highest fluorescence when tested in concentration of estradiol. Desiring the use of the most sensitive promoter, the same hybrid promoter was taken into this research, with the purpose of the STF binded with PFAS to attach to with high affinity.
Characterization
Labwork
Our plasmids were shipped to the lab at a concentration of 4 ng/ml. To start, A two step transformation protocol was used in order to transform E. coli with both plasmids three and four for our STF pathway. Both Kanamycin and Ampicillin antibiotics were utilized to screen for untransformed cultured bacteria.
These were transformed into DH5-alpha E. coli and cultured on Kanamycin plates containing X-gal. The presence of colonies and positive blue/white screening results indicate successful transformation.
Blue-White Screening Results:
We performed a restriction digest with SapI and ran the results on a gel. The bands that were produced were as expected. The ladder is 1 kb per band.
Gel Electrophoresis Results:
Once we confirmed proper gel results, we selected two colonies and grew them up. We then exposed them to PFOA and recorded the fluorescence in a fluorometer. The results of the fluorescence of our construct are shown below:
Graph 7 displays the fluorescence values over time of a colony that was taken from plate 1, containing E. coli taken from the Estrogen Receptor STF construct after subtracting the LB broth fluorescence values at the corresponding times. Since this graph doesn’t follow the previous and confirmed trend of fluorescence intensity increasing when PFAS (PFOA) is added, it can be hypothesized that when applied in real-time, this construct is less likely to be able to provide accurate results via fluorescence.
The data in Graph 8 implies that all cells produce a basal fluorescence over time based on the increasing fluorescence reading across all cells. The amount of fluorescence at any time point appears to be inversely related to PFAS concentration, however more testing is needed to determine if the ordering is statistically significant and not an artifact of any inaccuracies in the fluorimeter’s readings.
There may be several reasons why our fluorescence results came out inconclusive. One major reason may be that the PFAS chemicals may not bind correctly to the STF, which prevents its activation of expression on the hybrid promoter. Another reason may be that the conformational change does not correctly occur to activate expression on the hybrid promoter. In addition, there may be native key transcription factors that are necessary to induce the hybrid promoter that is only found in yeast cells and absent in E. coli, suggesting that there is significantly less transcription in our E. coli. The last contributing factor to our inconclusive results may be that the VP16 activator domain may not have functioned as desired in our E.coli. VP16 is native to herpes simplex virus proteins which mainly target eukaryotic cells. The RNA polymerase in eukaryotic cells is fundamentally different compared to prokaryotic cells, which means that the VP16 may not have successfully recruited the polymerases to the DNA as desired.
These reasons call for the need for future research on and testing to confirm the viability of the use of the estrogen receptor STF as a potential mechanism for biosensing of PFAS in E.coli.
Possible Uses for Other Teams
There may be several reasons why our fluorescence results came out inconclusive. One major reason may be that the PFAS chemicals may not bind correctly to the STF, which prevents its activation of expression on the hybrid promoter. Another reason may be that the conformational change does not correctly occur to activate expression on the hybrid promoter. In addition, there may be native key transcription factors that are necessary to induce the hybrid promoter that is only found in yeast cells and absent in E. coli, suggesting that there is significantly less transcription in our E. coli. The last contributing factor to our inconclusive results may be that the VP16 activator domain may not have functioned as desired in our E.coli. VP16 is native to herpes simplex virus proteins which mainly target eukaryotic cells. The RNA polymerase in eukaryotic cells is fundamentally different compared to prokaryotic cells, which means that the VP16 may not have successfully recruited the polymerases to the DNA as desired.
These reasons call for the need for future research on and testing to confirm the successability of the use of the estrogen receptor STF as a potential mechanism for biosensing of PFAS in E.coli.
Possible Uses for Other Teams
Our experiment was used to determine an approach to PFAS detection through the use of an estrogen receptor STF and a hybrid promoter inside of E. coli. There was no significant fluorescence difference between the different levels of PFAS. These results could have been due to human error or the possibility that PFAS does not bind well with the estrogen receptor, pushing the need for further research.
Although we didn’t see much success in the results of this protein within E. coli, other teams could use this part for either retesting to experiment whether the STF is capable of binding to the hybrid promoter.
More testing of the corrected sequence is needed to properly determine the real functionality of this promoter.
References
Dossani, Z. Y., Reider Apel, A., Szmidt-Middleton, H., Hillson, N. J., Deutsch, S., Keasling, J. D., & Mukhopadhyay, A. (2018, March). A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering. Yeast (Chichester, England). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873372/
ACS Publications: Chemistry journals, books, and references published ... (n.d.). http://pubs.acs.org/doi/full/10.1021/ja026939x?mobileUi=0
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