Paulsson:Fast matFP

From OpenWetWare

Jump to: navigation, search

Home        Group Meeting        Protocols        Inventory        Journal Watch        Top 100        Links        Facts of Life       


Contents

A project: faster maturing FPs

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

Thoughts

Personal tools