Part:BBa_K2957018
hEF1a, CCL5 with NeonGreen tag (C-terminal)
Contents: hEF1a, CCL5, NeonGreen tag, SynthpA, with Carb resistance
What is it?: A Composite part; it is a genetic circuit with hEF1a promoter and CCL5 gene allowing for constitutive CCL5 expression.
What does it do?: It codes for the chemokine, CCL5, which is a chemoattractant for T cells. It was important for our project in particular because of its involvement in the immune system. It has a NeonGreen tag attached.
How to use it?: This part was used for Type IIS Cloning and transfected into HEKs. We then were able to test for secretion of CCL5. See more on the Parts Overview Page or MIT iGEM 2019 wiki: https://2019.igem.org/Team:MIT/Parts.
Characterization
This was checked through sequencing and we have glycerol stocks of each of these.
The above graph shows our secretion test results, the main characterization of our parts (BBa_K2957018: hEF1a CCL5-NeonGreen and BBa_K2957007: hEF1a IL8-NeonGreen). To test whether our chemokine was successfully expressed and secreted, the culture team transfected HEK cells with IL8 and CCL5, both with NeonGreen fused to it as a fluorescent indicator. After performing a complete secretion test, we found a significant difference in fluorescent intensity for the CCL5 and the IL8 supernatant when compared to untransfected cells and cells transfected with just NeonGreen. This data suggests that our fusion proteins were translated and secreted properly as the fluorescent indicator was detected in the media outside of the cell. In addition, the transfected cells also had a large amount of protein production still inside of the cell as shown by the lysate. The fluorescence of the NeonGreen lysate is not completely accurate due to the intensity being too high for the reader to fully measure. Despite this, its high reading indicates that it was a proper positive control as it had significant fluorescence compared to the untransfected cells. The next challenges were then evaluating whether the chemokine protein was actually functional, which we detail our attempts to test so in the Results pages.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1224
Illegal PstI site found at 675 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 675
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 691
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1224
Illegal PstI site found at 675 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1224
Illegal PstI site found at 675
Illegal AgeI site found at 81
Illegal AgeI site found at 1230 - 1000COMPATIBLE WITH RFC[1000]
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