Team:MIT mammalian


Revision as of 08:03, 25 October 2010 by Ldeming (Talk | contribs)

The Cellular Touchpad
Our summer iGEM goal was to build a cellular touchpad, a cell line capable of responding to mechanical stimulus by differentiating into bone tissue. We split the project up into three parallel modules: mechanical sensing, signal processing and bone differentiation. For the mechanosensing portion of the project, we searched the literature for potential mechanosensitive promoters, then cloned them into expression vectors containing EGFP. We used plate shaking and microfluidic devices to mechanically stimulate the cells and screen for shear stress-responsive candidates. With signal processing, our goal was to convert a short pulse of mechanical stimulation into a permanent 'switch' for differentiation. We designed, built and tested a synthetic gene circuit controlled positive feedback of the rtTA3 transcription factor. The circuit showed robust upregulation after the activation of an inducible promoter.

For bone differentiation, we decided to create a dual cellular system. BMP2 (Bone Morphogenetic Protein) is a fast and efficient inducer of osteoblastogenesis; we plan to construct the cellular circuit in HEK cells, which are easier to engineer, and have them inducibly secrete BMP2 to differentiate co-cultured stem cells. We also managed to induce bone formation in two different stem cell lines, using human recombinant BMP2, and detect it on a western blot of cellular supernatant. In summary, we've accomplished what we set out to do - test mechanosensitive promoters, build a cellular circuit in mammalian cells, and induce bone differentiation in stem cells.

Along the way, we also managed to create a new assembly standard for mammalian cells. 'MammoBlock' is a recombination-based protocol (see our New Mammalian Standard page for more information), especially useful when dealing with long mammalian construct sequences. It's a robust and efficient cloning procedure, that allows for quick creation of high-quality entry vectors. Like our morphogenetic toolkit, it is meant to act as the groundwork for future expansion in the world of mammalian synthetic biology.