Difference between revisions of "Endy:Notebook/Gemini"
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<!-- sibboleth --><div id="lncal1" style="border:0px;"><div style="display:none;" id="id">lncal1</div><div style="display:none;" id="dtext">08/28/2010,09/14/2016</div><div style="display:none;" id="page">Endy:Notebook/Gemini</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month"></div><div style="display:none;" id="year"></div><div style="display:none;" id="readonly">Y</div></div>
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The goal is to highlight an operating regime in which Gemini is uniquely capable, meaning that either LacZ or GFP alone would be insufficient. It is hypothesized that the dual reporter is uniquely capable when both population and single cell measurements are desired, but gene expression is weak due to either a low copy plasmid and / or weak RBS and promoter. With this in mind, the picture to the left shows the utility of the reporter: it can report weakly expressed genes using the plate reader and LacZ where type GFP expression is undetectable. Yet, it can also be used for single cell analysis with GFP and microscopy, where wild type LacZ could not.
A GFP-LacZ fusion dual reporter has been built and shown to encapsulate the positive properties of LacZ and GFP Hwang, 2006. This dual reporter was designed for the study of mammalian cells, specifically cell lineage within the embryo and stem cells. With this in mind, is not optimally designed for use by the synthetic biology community for three reasons.
Evaluation of tetrameric lacZ structure shows N-termini of two adjacent lacZ alpha fragments in contact (or at the very least are in close proximity), whereas the C-termini point out into solution. Given this consideration, complimentation with C-terminal LacZa fusion to the N-terminal of GFP seems more likely to work than N-terminal LacZ fusion. That said, the first 12 residues of lacZ are not resolved in the structure. So, it may very well by that N-terminal additions are perfectly acceptable; we know from Hwang et al that LacZ monomers appear able to form a tetramer with GFP fused to the N-terminal.
Two designs utilizing the alpha fragment of beta-gal were built. The first is N-terminal of LacZa to C-terminal of GFP fusion (BBaE0050, shown left). This is similar to the design of Hwang et al (2006) except for the fact that it does not have a linker. This design did not work. A linker was also added to the C-terminal of LacZa fusion to N-terminal GFP (part BBaE0051, right). The linker is glycine/serine rich for flexibility with a few charged residues to aid in solubility. This design is characterized below.
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 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.
Key points to make
Within a certain, previously defined operating regime:
1. Wild type GFP is insufficient because it cannot be detected via plate reader
2. LacZ alone is insufficient because it cannot be detected via microscopy
3. Gemini produces a signal that can be detected by both mediums in domain in which either one would be insufficient
1. Show : wild type GFP is insufficient because it cannot be detected via plate reader
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.