Team:MIT/Basic Part
Basic Parts (pL0s)
Our basic parts are neatly divided below into the standard BioBrick categories so that we could form our parts collections: the Swarming Chemokine Toolbox collection and the synNotch Cell Communication collection. We call these parts pL0s according to MoClo Golden Gate Assembly (see Parts Overview page for more information) and these can be assembled into our composite parts (pL1s).
We used different backbones from iGEM’s Standard Biobrick of pSB1C3 in order for them to be Type IIS/Golden Gate compatible. With this system as well we are able to differentiate between our basic parts and composite parts, as well as whether they were correctly assembled (i.e. we used X-gal and spec for basic parts, and RFP and carb for composite parts). If you would like more information on this, please email us at igem-2019-students@mit.edu or the Weiss Lab for their cloning system protocols. In order to form our composite parts, we used Golden Gate Modular Cloning Assembly (see Parts Overview Page for a graphic of how we did this.
| 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_K2957011 | Coding | CCL5 (human) |
| BBa_K2957012 | Coding | CCL5 with Flag Tag |
| BBa_K2957013 | Coding | CCL5 with NeonGreen tag (C-terminal) |
| BBa_K2957043 | Regulatory | CMV-2xGal4 (Gal4 repressible promoter) |
| BBa_K2957057 | Regulatory | hEF1a |
| BBa_K2957058 | Regulatory | TRE |
| BBa_K2957059 | Regulatory | CMV |
| BBa_K2957061 | Reporter | mKate2 |
| BBa_K2957068 | Terminator | Synthetic PolyA |
| BBa_K2957077 | Backbone | Plasmid_Backbone pL1 position 1 backbone (ST1-2) |
| BBa_K2957094 | Coding | C5a |
| BBa_K2957095 | Coding | NeonGreen C5a |
| BBa_K2957096 | Coding | Flag-C5a |
We used different backbones from iGEM’s Standard Biobrick of pSB1C3 in order for them to be Type IIS/Golden Gate compatible. With this system as well we are able to differentiate between our basic parts and composite parts, as well as whether they were correctly assembled (i.e. we used X-gal and spec for basic parts, and RFP and carb for composite parts). If you would like more information on this, please email us at igem-2019-students@mit.edu or the Weiss Lab for their cloning system protocols.
In order to form our composite parts, we used Golden Gate Modular Cloning Assembly (see Parts Overview page for a graphic of how we did this.
Best Basic Part:
BBa_K2957043 - CMV-2xGal4 Promoter
The purpose of creating this part was to create a negative feedback system for our synNotch system should it be successful. Although we ultimately did not get to testing our synNotch system in practice, the CMV-2xGal4 promoter could potentially have future uses as a repressible promoter for other circuits future iGEMmers may decide to use. For this reason, we have chosen this as our best basic part over the other basic parts directly used for the composite parts of our collections.
Basic Part Characterization:
We also did further characterization on: BBa_K1119006 or BBa_I712004 (CMV Promoter), which is a CMV promoter in comparison to the hEF1a promoter, BBa_K2520023, already in the iGEM database. Specifically we characterized the differences we saw in terms of results after transfection of HL60s with circuits containing CMV vs. hEF1a. Parts Characterization