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Team:Berkeley/Project/Localization
From 2012.igem.org
To do this, we needed to decide which organelles to target and how to target them. We also had to optimize our choice of promoters so that one fluorescent protein signal was not too strong or too weak in comparison to the rest.
Nucleus.
Cellular Periphery-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.
Actin.
Vacuolar Membrane
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!
When choosing promoters for our cassettes, we had to find promoters that would make the MiCode's fluorescent proteins easily visible under the microscope. Furthermore, the promoters would have to adjust for differences in protein abundances and consequently the relative brightness for each localization tag. A portion of our project was to decide on this optimum set of promoters.
Out of the 7000+ promoters in the yeast genome, we decided to experiment with 5 promoters: pTEF1, pTDH3, pRPL18B, pRNR2, and pREV1 - listed from strongest to weakest. These were chosen based on the data from a paper from UCSF (Huh et al, 2003) in which every yeast genome open reading frame was tagged with GFP and observed for protein abundance. The 5 promoters listed above were simply 5 promoters that spanned a reasonable range in promoter strength and were promoters that Dueber lab was already familiar with. The promoter library we created allowed us to systematically compare all 5 promoters. Each yeast strain in the library expressed only one organelle localization tag, with one promoter, and we imaged them all under the same microscope settings. Two modes of image analysis were used for thorough comparison: one with the pixel brightness range set from 0-4095, and another with range 25-1000. The data can be seen here.
To assay their potential use in our project, we characterized the strength of several registry yeast promoters. We wanted to compare their consistency of expression and relative fluorescence against our lab's strongest promoter, pTDH3. The promoters we used in our assay:
- pSTE5 (weak)
- pCYC1 (medium)
- pADH1 (strong)
- pTDH3 (strongest)
Experimental Design: We designed our promoters to express a fluorescent protein so that we could quickly measure bulk fluorescence via TECAN. To roughly test if downstream sequence affects expression, we cloned the promoter in front of two different fluorescent proteins, yellow fluorescent Venus and red fluorescent mKate. The device was cloned on a backbone with Leu2 marker and Cen6 origin of replication.
After
