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. Author manuscript; available in PMC: 2012 Nov 18.
Published in final edited form as: ACS Chem Biol. 2011 Sep 8;6(11):1205–1213. doi: 10.1021/cb2001389

Figure 4. Degradation of LacI-DAS+4.

Figure 4

(A) Assay strains contained an sspB deletion and a DAS+4 tag at the C-terminus of LacI, which represses transcription of lacZ. Production of the split-adaptor components is mediated by plasmid-borne constitutive promoters. (B) Time course of degradation of LacI-DAS+4 (8 μM) by ClpX6 (0.3 μM), ClpP (0.9 μM), SspBCORE-FRB (5 μM), FKBP12-SspBXB (5 μM) and 10 μM rapamycin (when present). (C) β-galactosidase activities were measured in strains containing LacI-DAS+4 following addition of rapamycin (10 μM), IPTG (5 mM), or DMSO. An isogenic strain expressing wild-type LacI showed no response to rapamycin. In the absence of inducer, the LacI-DAS+4 strain exhibited increased basal β-galactosidase activity relative to the wild-type LacI strain. Further experiments indicated that the observed de-repression is not solely due to rapamycin-independent LacI-DAS+4 degradation or SspBCORE-FRB mediated inhibition of LacI activity (37).