A project: faster maturing FPs
P(regulated)-UBP1-[zFP] (on the chromosome?)
- expression levels must be such that R-xFP doesn't overwhelm clp machinery (ie undetectable in wt, detectable in clpx mutant)
Use UBIQ-L-xFP, instead of UBIQ-R-xFP
Adjust inducer (arabinose?)
Integrate UBP1, or keep on a plasmid?
growth rate (temperature, growth media)
fast shut off of Ubp1 expression to allow for detection... temperature sensitive ubp1?? screen for bright -Ubp1, then re-screen for bight +Ubp1
capacity of clp machinery (modulatable? above wt level?)
detection limit with FACS (other?), visualizing number of molecules that could be degraded
plasmid used in GFP screens? cell-to-cell variability?
a variety of degradation tags: ssrA, n-term R, n-term L, RpoS, RepA, HemA, LacI
- used in combination?
Compare in assay, two extant FPs (preferably yellow) that have been shown to have different maturation kinetics (approx 2 fold).
fraction matured, and observed for various maturation rates and degradation rates
multiple processes/rates to reach degradable state