Team:MIT/Part Collection
Parts Collections
Swarming Chemokine Collection
![](“https://static.igem.org/mediawiki/2019/7/7e/T--MIT--design-part.jpg")
The goal of this collection is to engineer what we call “leader” cells (specifically HEK cells for our experiments) with several constructs of interest that contain different promoters (hEF1a, CMV, TRE) and different chemokines (IL8, CCL5, C5a, etc.) involved in immune cell swarming behavior. Ultimately, we hope that this “swarming” chemokine collection will continue to be further expanded into a full immunology toolbox with more complex chemokines. Below is a table showing all of our basic parts by general name. To assemble our whole collection we used MoClo assembly as detailed on the Basic Parts page. The combination of all these parts resulted in a collection of around 24 constructs, with 21 constructs successfully made.
To see the full list of our parts that we planned to make or were successfully made, please see this link: Contact us at igem-2019-students@mit.edu to gain access to the sequence files!
The following table has our Swarming Chemokine parts. Pressing on a part will take you to the part page with more details.
| Name | Type | Description |
|---|---|---|
| BBa_K2957000 | Coding | IL-8 (CXCL8) |
| BBa_K2957001 | Coding | IL-8 (CXCL-8) with FLAG Tag |
| BBa_K2957002 | Coding | IL-8 (CXCL-8) with NeonGreen tag |
| 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_K2957011 | Coding | CCL5 (human) |
| BBa_K2957012 | Coding | CCL5 with Flag Tag |
| BBa_K2957013 | Coding | CCL5 with NeonGreen tag (C-terminal) |
| 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_K2957031 | Composite | TRE, CCL5 |
| BBa_K2957032 | Composite | TRE, CCL5-Flag |
| BBa_K2957033 | Composite | TRE, CCL5-NeonGreen |
| BBa_K2957034 | Composite | TRE, C5a-NeonGreen |
| BBa_K2957057 | Regulatory | hEF1a |
| BBa_K2957058 | Regulatory | TRE |
| BBa_K2957059 | Regulatory | CMV |
| BBa_K2957094 | Coding | C5a |
| BBa_K2957095 | Coding | NeonGreen C5a |
| BBa_K2957096 | Coding | Flag-C5a |
synNotch Cell Communication Collection
More than halfway through our project and after talking with Dr. Irimia and Dr. Mansour from Massachusetts General Hospital (MGH), we realized that IL8 expression by engineered cells would not necessarily be enough to generate significant neutrophil swarming. In fact not only was overexpression of IL8 necessary, but we were also told that leukotriene-B4 (LTB4) was actually a stronger candidate as a chemokine. However, LTB4 is a protein that is only synthesized following the expression of several enzymes, and so we could not perform LTB4 synthesis. Instead, our mentor Dr. Shiva Razavi suggested we pursue at this time a synNotch system to not only indicate cell-to-cell communication between our leader cells (HEKs) and follower cells (HL60s) but also to potentially increase the response of neutrophils by enhancing the signal created by IL8. We imagine that this will also simulate the second wave of neutrophil swarming that is often seen naturally that Dr. Irimia noted to us.
The goal of this collection is to create a system that will be able to perform the following:
Diagrams by Melody Wu, based on mentor Shiva Razavi’s diagrams
CD19 is a membrane receptor which when coming into contact with the receptor of another cell (the “sender”) will respond by releasing a TetR protein that will induce the expression of a chemokine/fluorescent protein of interest (positive feedback). We hope that this will upregulate the signal of the chemokine of interest, which can lead to an increased foundational understanding of the impacts of chemokine expression on immune cell behavior. However, we believe this system can become even more complex, with “follower” cells also becoming themselves sender cells that amplify the signal at the location of interest (as shown below).
The following table has our synNotch Cell Communication parts. Pressing on a part will take you to the part page with more details.
| Name | Type | Description |
|---|---|---|
| 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_K2957043 | Regulatory | CMV-2xGal4 (Gal4 repressible promoter) |
| 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 |
This page was written by Melody Wu