20151027Tue this "75"
is my update of "65" posting
See text at
for text summary. The PDf here in "75" supersedes "65".
The PDF here has 10 pages added to the end from the "65" version (pages 40-49 of PDF when including the bepress cover page)..
here is the text in my cover page (bepress may add its own cover):
20151027Tue: added 10 page e-poster at bottom explaining Binary Tattletales and T-Bow. That is, binary with respect to protein components. For one color (number of repeats, epitope tags, FPs are examples, here rounded to convenient numbers):
1. 100 copies of synthetic tandem repeat ("synTR").
2. 10 copies of epitope tag as TALE-(linker-epitope)10 ... see new page 48 (e-poster page 10) for coiled coil domains.
3. 10x brighter green FP by binder-(linker-bfloGFPa1)3.
At maximal loading (and no overexpression to pollute the cell with fluorescent smog), this would result in a one dimensional spot in a live cell of:
100 x 10 x 10 = 10,000 times brighter than a single EGFP molecule.
* synTR: (say) 16 base pairs with (say) eight bases (different between each repeat). If using Cas9, adjust parameters (length,
complement to PAM) to suit each orthogonal Cas9:guide RNA unit. I note that VNTRs of similar length exist in the human
genome, such as MUC1 (see Kirby ... Lander 2013 Nat Genet, for sequences), MUC4, telomerase, and more.
* TALE or Cas9 ... see also Tanenbaum 2014 "SunTags" paper, with 24 (early part) or 10 (later) epitope tags per Cas9.
* 10x brighter GFP than the 'standard of care' EGFP from 1996 (is anyone reading this still driving a 1996 minivan?).
bfloGFPa1 and mNeonGreen are ~3.x5 brighter than EGFP. For dimmer fluorescent proteins, say mKate2, add more FPs
and/or increase the number of units of the multimeric epitope tags. I suggest Coiled coil domains (molecular biologists may
know these better as "Leucine zippers", as in the domians that enable Jun and Fos to heterodimerize to form the AP-1
transcription factor), whose heptad repeats pairing rules are now well defined (or use "CC" from some distant organism that
will not pair with human, etc, coiled coils). 4 or 5 heptad repeats results in high (nM and very high (sub-nM) affinities. Plenty
of orthogonal pairs of "CC" heterodimers are known. Heterotrimers (both ABC and AAB) are known (Kiyokawa 2004
Chemistry) -- could make TALE-(linker-Coiled coil B)10 that would pair with TWO Coiled coil A-(linker-FP)n.
- Binary tattletales,
- Rainbow T-cells and Tumor cells,
- Rainbow multiplex transcriptional reporters; kick button on Dual glow luciferase assays
Available at: http://works.bepress.com/gmcnamara/75/