Part:BBa_K2957007
hEF1a, IL-8 (CXCL-8), NeonGreen Tag
Contents: hEF1a, IL-8 (CXCL-8), NeonGreen Tag, SynthpA, with Carb resistance
What is it?: A Composite part; it is a genetic circuit with hEF1a promoter and IL-8 gene allowing for constitutive IL-8 expression
What does it do?: It's responsible for the expression of the chemokine, IL-8 (CXCL8) with Flag tag. It was important for our project in particular because of its involvement in the immune system and is known to create neutrophil swarming.
How to use it?: This part was used for Type IIS Cloning and transfected into HEKs. It also has a NeonGreen tag attached to the IL8 so this allows us to visualize it and determine transfection efficiency. See more on the Parts Overview Page or MIT iGEM 2019 wiki: <a href=https://2019.igem.org/Team:MIT/Parts>."
Characterization
Our team’s best composite part is the hEF1a IL8-NeonGreen construct as we were able to prove it’s functionality in engineering our leader cells, HEK-293T, to secrete a major chemokine of interest to us, IL8, which is involved in inflammatory response and has been shown to lead to neutrophil swarming (secretion test results below). IL8 induces mast cells to produce histamines, which causes openings in the endothelium, which allows neutrophils to move through. We were also able to test the version of this plasmid (BBa_K2957003) with our follower cells, HL60s, in an ibidi chamber (see video on Results page of MIT iGEM 2019 wiki). This was checked through sequencing and we have glycerol stocks of each of these.
Secretion Test Results
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.
How are secretion test was performed is detailed below (as well as in our Results pages):
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1256
Illegal PstI site found at 699 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 699
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 473
Illegal BamHI site found at 715 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1256
Illegal PstI site found at 699 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1256
Illegal PstI site found at 699
Illegal AgeI site found at 81
Illegal AgeI site found at 1262 - 1000COMPATIBLE WITH RFC[1000]
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