Part:BBa_K3027002:Experience

Plasmid Description

pBAD24 contains an ampicillin resistance gene, a pBR322_origin for replication, an araC gene coding the AraC protein and a PBAD promoter. The Nuclease_yqcG gene was inserted in the pBAD24 plasmid under the control of PBAD. AraC regulates the expression of genes controlled by the PBAD. The presence of glucose in media inhibits the adenyl cyclase producing cAMP. cAMP forms a complex with the CRP protein to activate the transcription of araC gene. Therefore, when glucose is absent from media, synthesis of AraC is stimulated. This protein forms a homodimer, which represses the transcription of genes under the control of the PBAD promoter, in the absence of arabinose. Arabinose modifies the conformation of the homodimer AraC. This alteration activates the transcription of genes controlled by PBAD. This design allows a tight regulation of the inserted gene by glucose (repression) and arabinose (induction). We modified plasmid pBAD24 to introduce two BsaI sites downstream the PBAD promoter to facilitate cloning using the Golden Gate technique. This new plasmid, named pBAD24-MoClo, allows type IIs cloning with the prefix AATG and the suffix GCTT. This cloning strategy was used to clone nuclease yqcG gene, yielding the plasmid pBAD24-nuclease_yqcG.