Team:MIT/Composite Part



Composite Parts

The following table has our composite parts. Pressing on a part will take you to the part page with more details.


Name Type Description
BBa_K2957003 Composite hEF1a, IL-8 (CXCL-8)
BBa_K2957004 Composite CMV, IL-8 (CXCL-8)
BBa_K2957005 Composite hEF1a, IL-8 (CXCL-8), Flag Tag
BBa_K2957006 Composite CMV, IL-8 (CXCL-8), Flag Tag
BBa_K2957007 Composite hEF1a, IL-8 (CXCL-8), NeonGreen Tag
BBa_K2957008 Composite CMV, IL-8 with NeonGreen Tag (C-terminal)
BBa_K2957009 Composite hEF1a, NeonGreen-C5a
BBa_K2957010 Composite CMV, NeonGreen-C5a
BBa_K2957014 Composite hEF1a, CCL5
BBa_K2957015 Composite CMV, CCL5
BBa_K2957016 Composite hEF1a, CCL5-Flag
BBa_K2957017 Composite CMV, CCL5-Flag
BBa_K2957018 Composite hEF1a, CCL5 with NeonGreen tag (C-terminal)
BBa_K2957019 Composite CMV, CCL5 with NeonGreen tag (C-terminal)
BBa_K2957020 Composite TRE, IL-8 (CXCL8)
BBa_K2957021 Composite TRE, IL-8 (CXCL8) with Flag Tag
BBa_K2957022 Composite TRE, IL-8 (CXCL-8) with NeonGreen tag (C-terminal)
BBa_K2957023 Composite hEF1a, mKate2
BBa_K2957024 Composite CMV, mKate2
BBa_K2957025 Composite TRE, mKate2/td>
BBa_K2957028 Composite hEF1a, NeonGreen
BBa_K2957029 Composite CMV, NeonGreen
BBa_K2957030 Composite TRE, NeonGreen
BBa_K2957031 Composite TRE, CCL5
BBa_K2957032 Composite TRE, CCL5-Flag
BBa_K2957033 Composite TRE, CCL5-NeonGreen
BBa_K2957034 Composite TRE, C5a-NeonGreen
BBa_K2957035 Composite TRE-tight, mKate
BBa_K2957036 Composite hEF1a, synCD19
BBa_K2957037 Composite hEF1a, CD19
BBa_K2957038 Composite hEF1a, EYFP
BBa_K2957039 Composite hEF1a, mKate
BBa_K2957040 Composite CMV-2xGal4 (Gal4 repressible promoter), IL-8 (CXCL-8)
BBa_K2957041 Composite CMV-2xGal4 (Gal4 repressible promoter), IL-8(CXCL-8), Flag Tag
BBa_K2957042 Composite CMV-2xGal4 (Gal4 repressible promoter), IL-8 (CXCL-8), NeonGreen Tag
BBa_K2957045 Composite CMV-2xGal4 (Gal4 repressible promoter), CCL5
BBa_K2957046 Composite CMV-2xGal4 (Gal4 repressible promoter), CCL5, NeonGreen Tag
BBa_K2957047 Composite CMV-2xGal4 (Gal4 repressible promoter), mKate2
BBa_K2957048 Composite CMV-2xGal4 (Gal4 repressible promoter), EYFP
BBa_K2957049 Composite CMV-2xGal4 (Gal4 repressible promoter), TagBFP
BBa_K2957050 Composite CMV-2xGal4 (Gal4 repressible promoter), iRFP720
BBa_K2957051 Composite CMV-2xGal4 (Gal4 repressible promoter), NeonGreen

Favorite Parts/Validated Parts:

Those that were created with help from the Weiss Lab’s parts are * as such.
Those that were created with Basic Parts we created are ** as such.
Those that were created with iGEM registry basic parts are *** as such.
Those that are fluorescent protein circuits are **** as such and were primarily used as visual transfection controls and were used when we characterized our Basic part (CMV, BBa_K1119006) in comparison to iGEM Basic part (hEF1a, BBa_K2520023)

Best Composite Part: BBa_K2957007 (pIG_053)

BBa_K2957007 (pIG_053)


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).


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):

References

  • Baggiolini, Marco, and Ian Clark-Lewis. “Interleukin‐8, a Chemotactic and Inflammatory Cytokine.” FEBS Press, John Wiley & Sons, Ltd, 16 Jan. 2002, febs.onlinelibrary.wiley.com/doi/epdf/10.1016/0014-5793%2892%2980909-Z.