Paulsson:Facts

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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!!!

What we know

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. 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

• E. coli doubling times

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

• 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

• radius of photobleached area (when acquiring Matrix of images on scope, how far apart should frames be?)

can be limited with constriction of field diaphragm to field of view

• 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?