Part:BBa_K5114231
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 STF 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 STF consisted of three subunits: LexA, the DNA binding domain; hER, the estrogen receptor binding domain; VP16, the activator domain. By combining the three domains together, the aim was to successfully create the estrogen receptor STF. Since PFAS acts as an agonist for estrogen receptors, it was hypothesized that the STF developed to detect estradiol concentrations could be used interchangeably with PFAS for detection.
Usage and Biology
STF - LexA: The LexA gene is naturally found in E. coli. When expressed, LexA consists of two smaller sub-domains connected by a short linker. LexA plays a crucial role in the response when DNA is damaged. LexA binds to the DNA, fixing damaged DNA strands. LexA binds to a specific DNA sequence called the SOS box in the promoter regions of genes involved in DNA repair.
STF - hER: The human estrogen receptor (hER) is encoded by two main genes: ESR1 (alpha (ERα)) and ESR2 (beta (ERβ)), and is expressed in a variety of tissues, including the reproductive organs, breast, bones, cardiovascular system, and the brain. The estrogen receptor is activated by the binding of estrogen (a hormone); once bound, the receptor undergoes a conformational change, which allows it to bind to DNA sequences, known as estrogen response elements (EREs). The binding to the estrogen receptor can both repress and initiate specific genes. The binding domain of the estrogen receptor is known as the ligand binding domain, as it is the specific site where the receptor binds to estrogen (ligand).
STF - VP16: VP16 is a transcription factor of herpes simplex virus (HSV) type 1 that is involved in activating the viral immediate-early genes. VP16 does not bind directly to DNA. Essentially, this activator domain interacts with components of cellular transcription, such as the TATA-binding protein (TBP) and TFIID, to recruit RNA polymerase II to the promoter regions, upregulating transcription and translation of DNA. The C-terminal domain of VP16, known as the VP16 transactivation domain (TAD), is responsible for transcriptional activation.
Essentially, when estrogen STF interacts with PFAS, the STF attaches to the pLex promoter, initiating transcription of the downstream GFP gene. The synthetic transcription factor has been designed to specifically recognize pLex through its domain LexA and VP16, LexA having highest affinity to the hybrid promoter and VP16 enhancing attachment. The use of the STF to induce expression of GFP is theoretically controlled only by the presence and activity of the synthetic transcription bounded with PFAS.
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.
To confirm transformation of both plasmids three and four, a blue-white screening and gel electrophoresis were utilized. The results of the Blue-White screening and Gel Electrophoresis are shown below:
Blue-White Screening Results:
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 viability of the use of the estrogen receptor STF as a potential mechanism for biosensing of PFAS in E.coli.
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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