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. 2018 Nov 29;14(11):e8605. doi: 10.15252/msb.20188605

Figure 3. Eliminating protein activity using a combination of CRISPRi, sRNA, and proteases.

Figure 3

  1. Schematic showing three levels of repression. A small guide RNA (sgRNA) directs deactivated Cas9 (dCas9) to block transcription from a promoter. sRNA binds to the mRNA and promotes degradation by recruiting Hfq. The mf‐Lon protease targets a tag (blue) added to the protein. The protease is also targeted to itself to reduce toxicity (Materials and Methods).
  2. Reduction of fluorescence of RFP by the different mechanisms of repression after 18 h of growth (Materials and Methods). The inducers are either 1 mM IPTG (sRNA) or 25 μM DAPG (sgRNA, mf‐LON). sgRNA and mf‐LON are co‐transcribed on a single transcript that is processed by ribozymes (Appendix Fig S13).
  3. The dynamics of repression by each of the mechanisms is shown. Empty circles are uninduced and black circles are induced (1 mM IPTG or 25 μM DAPG) at the 2‐h time point (dashed line; Materials and Methods).
  4. Metabolic pathways to acetate in Escherichia coli are shown along with the targeted enzymes. The impact of either knocking out these enzymes or knocking them down by the various mechanisms after 20 h is shown. On the left of the graph are shown empty strains containing either no acetate pathway modifications (WT; MG1655∆glnL), a double deletion (∆pta∆poxB), or single knockouts and protease tag modifications (∆poxB pta::pdt3, ∆pta poxB::E170). On the right are shown knockdowns of pta (blue) and poxB (red), tested in strains ∆poxB pta::pdt3 and ∆pta poxB::E170, respectively. Knockdowns are generated by expression of sgRNA, proteases, or both mechanisms encoded on a single transcript.
  5. Design of a PoxB mutant that can be targeted by the SuMMV protease. The structure of PoxB is shown (PDB: 3ey9; Neumann et al, 2008), highlighting the amino acid sites selected for degron insertion (in red). The impact on acetate production of different acetate pathway mutants is shown on the left of the graph. On the right of the graph, a double knockout mutant (ΔptaΔpoxB) contains variants of poxB expressed from a BAC. Note that complementation of unmodified poxB (WT) from the BAC does not fully reconstitute acetate production. N‐ter and E170 refer to the location of the SuMMV degron tag in poxB. A plasmid expressing protease SuMMV from a DAPG‐inducible promoter was also introduced into strains containing poxB variants WT and E170 complemented on a BAC and a strain poxB with an E170 site replacement on the genome (poxB::E170). The graph shows the acetate produced in these strains when either no inducer (white) or 25 μM DAPG (grey) is added to the culture.
Data information: Representative cytometry fluorescence distributions for (B, C) are shown in Appendix Fig S11. Error bars represent one standard deviation of three biological replicates done on different days. Bars with single (*) and double (**) asterisks indicate a statistically significant difference with P‐values < 0.01 and 0.001, respectively, as assessed by an unpaired t‐test.