Paulsson:Facts

The facts of life
A place for all those little things that come in handy...

Please provide a citation, credible source, or oath of authenticity to each FACT OF LIFE. Or better yet, a link to the data!!!

Fluorescent Proteins

 * in vivo maturation of Venus ~7 -/+ 2.5 min (Yu and Xie, Science'06)
 * homoaffinity of purified recombinant YFP, K(d) = 0.11mM (Zacharias et al., Science v296:913-916, 2002)
 * loops in GFP that accept insertions without loss of function (fluorescence):
 * 1) aa144-145 (Karmella Haynes)

Protein turnover

 * degradation of GFP-LAA: in vivo??? in vitro <5min (Bukau, Sue Wickner)
 * degradation of N-end rule substrate ~2min (Varshavsky, Science'91)

E.coli
strain W1485: 46.9' at 28deg, 24.1' at 37deg, 21.4' at 42deg (Condon and Squires, J.Bact 1995)
 * E. coli doubling times


 * supplementing minimal media with 0.1% casamino acids attenuates GFP toxicity (Alper and Stephanopoulos, PNAS'05)


 * Compared to closed circular dsDNA, nicked circular DNA and ssDNA are 50% as efficient, linear DNA is 0.1% as efficient in transformation of E. coli.

Lab Strains

 * The plasmid in ST11 and ST12 (and all pPM derivatives) have an A to G mutation in the PLtet-O1 promoter (at position 1982 in pPM5). This substitution doesn't appear to alter behavior of Ptet, but I haven't done the direct comparison to original PLtet-O1.


 * pPM1, pPM2 and derivatives have a "T7 promoter" flanking the MCS. This T7 promoter sequence is truncated and doesn't support expression by T7 polymerase, although it can be useful for sequencing.


 * PMB14 and 27 both contain integrated version of LacI AND TetR (at attB, phage lambda attachment site). They work equally well at repressing Plac and Ptet.

Microscopy

 * 0.3 um depth of field with 100x objective (Shay's estimate)
 * 80 micron x 80 micron Field of View on the CCD with 100x objective on the Zeiss scope (Shay's estimate, entry by Burak Aug 13, 08)
 * 120 micron x 120 micron Field of View on the CCD with 60x objective on the Zeiss scope (Shay's estimate, entry by Burak Aug 13, 08)

What we'd like to know
can be limited with constriction of field diaphragm to field of view
 * radius of photobleached area (when acquiring Matrix of images on scope, how far apart should frames be?)
 * how fast can you spin coli without losing cfu? stationary vs. log phase growth
 * percent of MFG-GFP fusions that are non-functional (eg in S.c. ORFeome collection)??? Partial loss of function may not be detected in viability, but rather in growth rate or protein localization?