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.