Part:BBa_K5535001
EHMT2, CMV Enhancer
This part is designed to mimic our other part (BBa_K5535000) in order to study addiction, but more so applicable to the preliminary rounds, and backed up with more evidence. We went with this plasmid to test and transfect into our cell line after extracting and growing it in culture, primarily due to the fact that it has the EHMT2 gene with a CMV enhancer that works to over-express our gene of interest. This plasmid also contains a V5 tag which is similar to the other plasmid structure we made, and we have proved that there is a clear difference between transfecting a cell with and without this plasmid.
OVERVIEW
We initially aimed to design a plasmid which was constructed to contain a CMV enhancer and the EHMT2 protein to increase production in cell lines. We sourced the plasmid’s sequence through gene synthesis, obtained the target gene, and transformed it into E-coli DH5-α. After, we transfected the said plasmid into HEK293 cell lines and ran the transfection through RT-PCR to detect the RNA levels in the cells after 4 days.
We validated our plasmid first after the extraction, getting a sequence back of 100% alignment.
TESTING
For our test, we transfected the said plasmid into HEK293T cells using Lipofectamine 3000 from Thermofisher, and we compared these results after a few days with cells that were not given the treatment with the plasmids transfected through qPCR. We expected to see a higher Cq value for the cells that were not given the plasmid, as it would have taken longer for them to amplify compared to those given a plasmid meant to overexpress this protein. The amount of cells we put in both wells were 1x10^5, and were left for 3 days' time before running the analysis.
The primers and probes we selected are as follows for our qPCR run:
EHMT2 qPCR forward primer: TGACTGCGTGCTGTTATTCC
EHMT2 qPCR reverse primer: GAGCTTGCGGTTGAGTTGAA
EHMT2 qPCR probe: CCGCAGCTCAGGGTTGGCCCCACGT
GAPDH qPCR forward primer: TTGTCAAGCTCATTTCCTGGTAT
GAPDH qPCR reverse primer: AGGGTCTCTCTCTTCCTCTTGT
GAPDH qPCR probe: AGACCCCTGGACCACCAGCCCCAGCA
The results of our test was shown below, comparing the EHMT2 expression of transfected vs non transfected cells. We used Cq values that told us the cycle number in which the gene amplified.
Note: The black line on the right hand side is the line we are comparing for EHMT2. When referring to cells, we mean cells per well in a 24 well plate.
0.5 x 10^5 cells, Treatment; transfected 3 days: Cq value of 21.41
0.5 x 10^5 cells, Control; left for 3 days: Cq value of 24.88
The graph of our Cq value comparison is shown below:
We can conclude that this plasmid worked on the mammalian cell line due to its lower Cq value with a difference of approximately 5, and therefore validate this plasmid's construct and function.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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