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A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia.
Hogan AM, Rahman ASMZ, Lightly TJ, Cardona ST
ACS Synth Biol. 2019 Sep 6. doi: 10.1021/acssynbio.9b00232.
PubMed Article

Plasmids from Article

ID Plasmid Purpose  
129389pAH25-SceIDerived from pDAI-SceI. Tetracycline resistance removed and replaced with chloramphenicol resistance cassette from pKD3.
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129390pAH-CTX1-rhaDerived from mini-CTX1. An integrase vector that can integrate the rhamnose-inducible promoter system into the chromosome of a host bacterium.
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129391pAH-CTX1-rhadCas9Derived from pAH-CTX1-rha. The Burkholderia cenocepacia codon-optimized dCas9 cloned downstream of PrhaBAD. Hence, dCas9 is controlled by the rhamnose-inducible promoter system.
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129392pAH-CTX1-rhadCas9-nativeDerived from pAH-CTX1-rha. The native Streptococcus pyogenes dCas9 gene cloned downstream of PrhaBAD. Non-codon-optimized dCas9 version of pAH-CTX1-rhadCas9.
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129463pSCB2-sgRNATemplate plasmid for guide sequence insertion via inverse PCR. Derived from pSCrhaB2-gRNA (see paper).
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129464pgRNA-guidelessDerived from pSCB2-sgRNA. 20 nucleotide binding sequence of sgRNA removed by inverse PCR. Used as a negative control.
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129465pgRNA-non-targetDerived from pSCB2-sgRNA. 20 nucleotide sequence added as sgRNA binding region by inverse PCR. Used as a negative control.
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154397pAH18Derived from pGPI-SceI; Trimethoprim resistant; carries I-SceI gene; contains 475 bp from both upstream and downstream flanking regions between the FRT sites on pAHCTX1-rhadCas9
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