Team:MIT/Mammalian synthetic biology kit

• BACKGROUND
• EXPERIMENTS
  Synthetic Promoters microRNA Recombinase Proof of Concept Collaborations
• FUTURE WORK
  Circuit Design Clinical Applications
• HUMAN PRACTICES
  Parts Toolbox Accessible Protocols Integrated Human Practices Public Outreach Interlab Study
• NOTEBOOK
  Lab Notebook Parts Mammalian Parts Collection Attributions Safety
• AWARDS
  HP Silver HP Gold Integrated HP Basic Part
• TEAM

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.

There are two plasmids involved:

pENTR: the entry plasmids contain the desired genes, flanked by L1 and L2 sites.

pDEST: the destination plasmid will eventually be transformed into the E.Coli. There is a selection or screening tool flanked by R1 and R2 sites.

From recombination of the L1, L2, R1, and R2 sites, the pEXPR expression plasmid is formed.

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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.

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 is a 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