Team:MIT/Part Collection

• 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

Mammalian Synthetic Biology Parts
Starter Kit

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. With more work, we can come up with better, faster, less invasive diagnostics, and if these systems are refined, we could potentially apply the same genetic circuit techniques to the field of treatments.

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 R2 and R4 sites.

From recombination of the L4, R1, L1, L2, R4 and R2 sites, the pEXPR expression plasmid is formed.

Currently, the selection tool used for the pDEST is ccdB. However, a patent on it, giving Invitrogen the proprietor of ccdB as a selection tool makes it impossible to distribute to iGEM teams. Because of this, iGEM had to discontinue multiple parts containing ccdB in their registry, and to avoid legal issues:

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.

Through an LR reaction, the pDEST mCherry combines with the pENTR gene and promoter plasmids. In the process, the mCherry is cut out. When eColi are transformed with the resulting plasmids and plated, the ones containing desired plasmids are white, while unsuccessful colonies fluoresce