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Team:Berkeley/Project/Localization
From 2012.igem.org
By localizing fluorescent proteins to specific organelles, each cell can be given a "microscopic barcode", or MiCode. Below you can see a single sample MiCode. Each member of a library will get a unique MiCode, distinguishing it from the rest of the library and tying the MiCode phenotype to a specific genotype.
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.
We chose four organelles from a list of over ten candidates based on the following criteria: (1) there existed targeting sequences in the literature, and (2) this organelle was visibly distinct from the other chosen organelles. The following were our favorites!
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
We used RFP, GFP, and CFP and our four organelles that could be easily distinguished: the nucleus, vacuolar membrane, cellular periphery, and actin. 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 unique MiCodes is 4,096!
When choosing promoters for our cassettes, we had to find promoters that would make each element of the MiCode 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
