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A) dCas9 was expressed from the chromosome of K56-2 under a rhamnose inducible promoter. Cells were grown to OD 600nm of 0.6 then induced with rhamnose for three hours. The soluble protein fraction was run on an 8% SDS gel. dCas9 was detected by an α-dCas9 antibody followed by a second antibody linked to alkaline phosphatase. The lane labelled Cas9+ was loaded with 5 ng of purified Cas9. B, C and D) Growth curves of B. cenocepacia K56-2∷dCas9 (B), dCas9 expressed with a non-genome <t>targeting</t> <t>sgRNA</t> <t>(dCas9/pgRNA-non</t> target) (C) and K56-2∷CTX1-rha, the vector control plasmid for the integration (D) in LB media show that expression of the chromosomally-encoded dCas9 induced with rhamnose up to 1% does not affect growth.
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A) dCas9 was expressed from the chromosome of K56-2 under a rhamnose inducible promoter. Cells were grown to OD 600nm of 0.6 then induced with rhamnose for three hours. The soluble protein fraction was run on an 8% SDS gel. dCas9 was detected by an α-dCas9 antibody followed by a second antibody linked to alkaline phosphatase. The lane labelled Cas9+ was loaded with 5 ng of purified Cas9. B, C and D) Growth curves of B. cenocepacia K56-2∷dCas9 (B), dCas9 expressed with a non-genome targeting sgRNA (dCas9/pgRNA-non target) (C) and K56-2∷CTX1-rha, the vector control plasmid for the integration (D) in LB media show that expression of the chromosomally-encoded dCas9 induced with rhamnose up to 1% does not affect growth.

Journal: bioRxiv

Article Title: A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

doi: 10.1101/618413

Figure Lengend Snippet: A) dCas9 was expressed from the chromosome of K56-2 under a rhamnose inducible promoter. Cells were grown to OD 600nm of 0.6 then induced with rhamnose for three hours. The soluble protein fraction was run on an 8% SDS gel. dCas9 was detected by an α-dCas9 antibody followed by a second antibody linked to alkaline phosphatase. The lane labelled Cas9+ was loaded with 5 ng of purified Cas9. B, C and D) Growth curves of B. cenocepacia K56-2∷dCas9 (B), dCas9 expressed with a non-genome targeting sgRNA (dCas9/pgRNA-non target) (C) and K56-2∷CTX1-rha, the vector control plasmid for the integration (D) in LB media show that expression of the chromosomally-encoded dCas9 induced with rhamnose up to 1% does not affect growth.

Article Snippet: The following plasmids have been deposited to Addgene for distribution: pAH-CTX1-rha (#129390), pAH-CTX1-rhadCas9 (#129391), pAH-CTX1-rhadCas9-native (#129392), pAH25-SceI (#129389), pSCB2-sgRNA (#129463), pgRNA-guideless (#129464), and pgRNA-non-target (#129465).

Techniques: SDS-Gel, Purification, Plasmid Preparation, Expressing