Paulsson:Facts: Difference between revisions
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* in vivo maturation of Venus ~7 -/+ 2.5 min (Yu and Xie, Science'06) | * 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) | * 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):<BR> | |||
## aa144-145 (Karmella Haynes) | |||
===Protein turnover=== | ===Protein turnover=== | ||
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*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. | *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. | ||
*PMB14 and 27 both contain integrated version of LacI AND TetR. They work equally well at repressing Plac and Ptet. | *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== | ==What we'd like to know== | ||
* radius of photobleached area (when acquiring Matrix of images on scope, how far apart should frames be?) | * 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 | * 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? | * 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? | ||
Latest revision as of 15:12, 28 May 2009
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):
- 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?
