With the development of efficacious gene editing tools and improved practices, mammalian synthetic biology grows more prevalent. While our project is one example of how synthetic biology can be used to diagnose diseases, we hope that more people are able to build off our work, and the work of others to apply synthetic biology to mammalian cells.
Hoping to see what amazing work can come from future teams and scientists, we have put together a toolbox of parts to make it easier to start mammalian work.
Our kit is meant to be used with Gateway cloning reactions. Gateway is a recombinase-based cloning method used by the phage λ against e.Coli, and isolated and optimized for for synthetic biology by Invitrogen.
![](https://static.igem.org/mediawiki/2016/3/34/T--MIT--gatewayrecombination2016.png)
There are two plasmids involved:
From recombination of the L1, L2, R1, and R2 sites, the pEXPR expression plasmid is formed.
![](https://static.igem.org/mediawiki/2016/f/fa/T--MIT--gatewayrecombined2016.png )
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Currently, the screening tool used for the pDEST is ccdb. However, a patent on it makes it impossible to distribute to iGEM teams. To allow distribution, we have created a pDEST plasmid that has mCherry fluorescence as a screening tool.
![](https://static.igem.org/mediawiki/2016/7/73/T--MIT--pdestmcherryplasmid.png)
Along with our pDEST, we include several pENTR vectors, with promoters and useful genes that can be combined and put into eColi to grow up before transfection into mammalian systems
hef1a: constitutive human promoter.
BM3R1, TALER_14, and TALER_21 are proteins that bind to their respective promoters and repress transcription
As repressible systems, we are including the DOX-inducible system, with a TRE (Tet Responsive Element) promoter, and SOMETHING.
The fluorescent markers available are eYFP, tagBFP, mKate, and mCherry
Directly from our project, we are including the promotors that we made: SOMETHINGSOMETHING
![](https://static.igem.org/mediawiki/2016/8/86/T--MIT--hef1atagbfp.png)
![](https://static.igem.org/mediawiki/2016/9/92/T--MIT--hef1mkate.png)
![](https://static.igem.org/mediawiki/2016/b/b4/T--MIT--hef1aeyfp.png)