Difference between revisions of "Endy:Notebook/Gemini"

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(Key points to make)
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1. Show : wild type GFP is insufficient because it cannot be detected via plate reader
* Confirm wild type GFP cannot be detected on the plate reader for weak promoters
** Therefore, we are getting '''controls''' for GFP built.
2. Show : Gemini produces a signal that can be detected by both mediums in domain in which either one would be insufficient
* LacZ activity is detectable via plate reader when GFP is not
** Need to demonstrate that LacZ is detectable (with MUG assay) and GFP is not (with '''control''')
*** Need to optimize MUG assay: protocol, cell growth / OD
* Over the same regulatory domain, GFP still is detectable via microscopy and LacZ is obviously not
** Need to show that GFP is indeed detectable via microscopy
3. Additional benchmarking : test Gemini against wild type LacZa and LacZ
* I don't think a promoter domain is necessary for this 
4. Sequencing :
* Optimize prep for low copy plasmid
* Use VF2 primer

Revision as of 13:41, 16 July 2009

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Florescence measurements with plate reader
Comparative measurements between two inoculations

Data follow the expect trend: florescence decreases with respect to promoter strength. For promoters 115, 113, and 112 florescence is barely detectable on the plate reader relative to the controls, and there is a substantial decline is signal strength below the strongest promoter.



MUG assay

MUG assay indicates beta-gal activity is identical for the three strongest promoters. Xgal plates show that 1) MUG assay correctly indicates beta-gal activity for the three strongest promoters (J23119, 101, and 106), 2) output for the moderately strong promoter J23115 is indistinguishable from from 119, 101, and 106 via Xgal plates, but is less detectable via MUG 3) J23113 and 112 are consistently undetectable. This is reasonable, as the activity of 112 and 113 are 1 and 21 au respectively, relative to 387 for 115, 1185 for 106, 1791 for 101, with all measured relative to 119, the strongest family member Promoter family.



It was designed by Joey Davis and Austin Che at MIT. The promoter-RBS variants used to generate a transfer function for Gemini were built by Austin Che and Justin Buck at MIT. The controls for benchmarking performance of Gemini are being constructed by Ginko Bio-works.

Possible extension

A better design, maybe? Maybe not.