Team:MIT mammalian Circuit

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Our project began with idea of a biological touchscreen. We envisioned a cellular 'iPad', a plate of cells that could sense applied pressure and differentiate in response. There are a ton of applications for this technology; at the most basic level, one could imagine drawing a pattern onto a cellular monolayer and watch bone form around the outline. The system could also be used to study morphogenesis, to explore the role of chemical and mechanical signaling in differentiation by trying to build analogous synthetic counterparts. We've developed a basic standard for linking mechanical sensing to cellular differentiation, a standard we hope to see it developed to support even more intricate systems.  
Our project began with idea of a biological touchscreen. We envisioned a cellular 'iPad', a plate of cells that could sense applied pressure and differentiate in response. There are a ton of applications for this technology; at the most basic level, one could imagine drawing a pattern onto a cellular monolayer and watch bone form around the outline. The system could also be used to study morphogenesis, to explore the role of chemical and mechanical signaling in differentiation by trying to build analogous synthetic counterparts. We've developed a basic standard for linking mechanical sensing to cellular differentiation, a standard we hope to see it developed to support even more intricate systems.  
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Our specific system involves cells that can sense mechanical stimulation and differentiate into bone in response. We broke our project up into three 'modules' to proceed in parallel. The first was a search for mechanosensitive promoters. The second focused on getting stem cells to differentiate into osteoblasts (bone-producing cells). The last module centered around creating a 'switch' in the mammalian cells that would allow us to convert a transient pressure pulse into long term expression of a differentiation program.  
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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.  
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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.
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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.
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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.  
<b>Click on the panels below to go to the experimental pages</b>
<b>Click on the panels below to go to the experimental pages</b>
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Revision as of 21:15, 26 October 2010

The Cellular Touchpad
Our project began with idea of a biological touchscreen. We envisioned a cellular 'iPad', a plate of cells that could sense applied pressure and differentiate in response. There are a ton of applications for this technology; at the most basic level, one could imagine drawing a pattern onto a cellular monolayer and watch bone form around the outline. The system could also be used to study morphogenesis, to explore the role of chemical and mechanical signaling in differentiation by trying to build analogous synthetic counterparts. We've developed a basic standard for linking mechanical sensing to cellular differentiation, a standard we hope to see it developed to support even more intricate systems.

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. Click on the panels below to go to the experimental pages

  Mechanical Signaling                  Circuit Design               Bone Differentiation

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