Part:BBa_M36091:Experience

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Results

Experiment 1: Transform construct into E. coli and make freezer stocks. Our streaking was successful, with clear colony growth. The plate was used to pick colonies for Miniprep DNA extraction and also for glycerol stock creation.

Experiment 2: PCR to confirm DNA construction PCR and Gel electrophoresis, using pGSH and primers designed around the PAX8 sequence and the TG promoter revealed an amplicon of length ~1300 base pairs. The PAX8 gene is 1371 base pairs long. This result suggests that no major construction errors occurred by confirming that the PAX8 gene of correct length is present in pGSH. Our negative control shows no band, as expected, telling us that there is no contamination or unexpected amplification occurring. . (Note: pComet results are explained later on, under the RT-PCR experiments.)

Experiment 3: Plasmid Transfection Success and Construct Functionality Assessment In the construct functionality experiment, high levels of GFP fluorescence in both pComet transfection control wells (glutathione at 0nM and 1mM), observed with fluorescence microscopy, suggest that plasmid transfection was successful and not inhibited by experimentally relevant glutathione concentrations. (Figure 3). The lack of signal in the negative control confirms that there is no unexpected signal observed. Non-transfected cells show no GFP signal, which is as expected. Experimental wells show extremely low levels of fluorescence. This does not provide conclusive proof that our construct worked, because the signal is too low to be sure. Further testing is required.

Experiment 4: Cell viability in Glutathione It was qualitatively observed, based on the light microscopy images shown above, that increasing concentrations of glutathione decreased cell viability. pComet wells and no transfection wells functioned as controls here also. They provided baselines against which to assess whether cell death appeared to be increasing compared to normal or decreasing compared to normal.

Experiment 5: RT-PCR to assess PAX8 and GFP transcription The RT-PCR experimental control was the pComet RT-PCR. We had clear results from microscopy in that well, so if the RT-PCR was working, we would expect to see a clear band in the pComet lane, and no band in the negative control (indicating no contamination). Looking back at Figure 3, this is exactly what was observed. This means that our experiment is working as expected, and also that the pComet is showing high levels of GFP and minimal primer contamination, which corresponds with the results from the microscopy. This means that the rest of our results can be interpreted knowing that the experimental setup is correct, and it provides a confirmation for the control pComet images shown in Figure 4. The results of our RT-PCR experiment for the negative controls functioned as expected. Though we see low levels of contamination, those lanes appear essentially clear, as expected. Furthermore, our GFP cell negative control (which is a lane with no construct transfection, but with DNA) shows what is probably a contaminant, but does not show GFP since the band is too high. That is as expected. We should not see GFP expression if there has been no transfection. Both PAX8 and GFP mRNA transcripts are being produced nearly identically in HeLa cells transfected with pGSH. This result was found regardless of the addition of additional glutathione. For PAX8, this shows that the constitutive promoter works as expected. Expression should occur, regardless of dosing. The expression of GFP in wells with and without additional glutathione, may be explained by two possibilities. The first possibility is that, the promoter sequence paired with GFP has a baseline level of expression that is independent of the activation of PAX8 by glutathione. The second possibility is that the wild type intracellular concentration of glutathione in HeLa is sufficient to activate PAX8 and induce expression of GFP. Since RT-PCR was only performed on wells with the lowest levels of glutathione, it cannot be concluded whether or not PAX8 transcriptional regulation of TG is being activated by glutathione. One rather surprising result that the PAX8 cell(-) lane also shows similar expression patterns. This lane represents a lane where DNA was loaded, but the DNA did not contain any construct DNA since those wells were never transfected. Therefore, we would have expected to see no PAX8 expression. The expression of PAX8 can be explained by the fact that HeLa cells have been shown to have low levels of PAX8 expression (18). However, there is some debate about the actual level of PAX8 expression in HeLa, so further work would be required to ascertain what is causing this result.

Applications of BBa_M36091

Sensing kidney transplant viability by measuring glutathione concentration.

---Glycerol Stocks Information---

Stanford Shriram Center for Bioengineering and Chemical Engineering

Barcode Numbers: ---0133024652 ---0133023846 ---0133026153

plasmid name: "CJMJPDSB"

antibiotic resistance: ampicillin

DNA 2.0 gene #193522

Expressed in HeLa cells

Glutathione Sensor (turns on green fluorescence)

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