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Team:Berkeley/Project/Localization
From 2012.igem.org
We targeted GFP to the cell periphery using a signal sequence derived from the Ras protein, a signal protein located in the cell membrane that activates in response to extracellular signals for growth and differentiation. HOW WE CHOSE THE ORGANELLES, AND HOW THEY LOOK.
The easiest way to create a barcode is by utilizing fluorescent proteins and seeing if the cell glows or does not glow. This creates a simple binary system. This can be expanded upon by using multiple fluorescent proteins. In our case, we used RFP, GFP, and CFP which can be expressed individually within the cell or in combinations with each other. At this stage, the number of MiCodes is represented by the function 2^x, x representing the number of distinct fluorophores. With three in use, our number of barcodes equalled 2^3 or 8 members. However, to match the size of common libraries used today in synthetic biology, we needed to expand our barcode system. Utilizing the power of microscopy to harness spatial information within the cell, we thought to target the fluorescent proteins to subcellular locations in the cell, the yeast's organelles. We chose four organelles that could be easily visually distinguished: the nucleus, vacuolar membrane, cellular periphery, and actin. Now our number of MiCodes was represented by the function 2^x^y where x still represents the number of fluorescent proteins while y represents the number of organelles to which the fluorophores are targeted. With three fluorescent proteins and four organelles, the number of MiCodes was increased exponentially from 8 to 4,096!
