FIG 2.
ClpXP-based controllable protein degradation system. (A) Top, diagram of the VSV-G-DAS4 tag integration vector and its use in tagging RNase E with a VSV-G epitope and DAS4 depletion tag. GentR, gentamicin resistant. Bottom, schematic representation of the ClpXP-based protein depletion system. Degradation of the RNase E-VDAS4 protein is depicted. (B) Western blot showing that RNase E depletion is dependent on sspB expression. V corresponds to the VSV-G epitope tag. The DAS4 tag allows for depletion of RNase E-VDAS4 in a manner dependent upon the intracellular concentration of the SspB adapter protein that directs RNase E-VDAS4 to the ClpXP protease complex. SspB synthesis is under the control of an IPTG-inducible promoter. FRT, FLP recombinase.