20151027Tue this "75"

http://works.bepress.com/gmcnamara/75

is my update of "65" posting

See text at

http://works.bepress.com/gmcnamara/65/

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.

Notes:

* 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.