Paulsson:Fast matFP

Assay overview
P(regulated)-UBP1-[zFP] (on the chromosome?)

P(constitutive)-UBIQ-R-xFP
 * expression levels must be such that R-xFP doesn't overwhelm clp machinery (ie undetectable in wt, detectable in clpx mutant)

Tweaking conditions
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

Important considerations
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?

Alternate approaches
clp mutants a variety of degradation tags: ssrA, n-term R, n-term L, RpoS, RepA, HemA, LacI
 * used in combination?

Test case
Compare in assay, two extant FPs (preferably yellow) that have been shown to have different maturation kinetics (approx 2 fold).

Expected distributions
fraction matured, and observed for various maturation rates and degradation rates multiple processes/rates to reach degradable state