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).
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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