Part:BBa_K3512001:Experience

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Applications of BBa_K3512001

Literature 1 Toxicity and antitoxicity plate assays To test the toxicity of the cloned CcdBO157 and CcdBF variants, the corresponding pBAD33-ccdB constructs were transformed in MG1655. The resulting transformants were plated on LB plates containing chloramphenicol with or without arabinose (1%). The CcdB variants were considered to be functional (toxic) when transformants were able to grow only in the absence of arabinose. To test the ability of the cloned CcdAO157 and CcdAF variantsto counteract the toxicity of CcdBO157 and CcdBF, respectively,the corresponding pKK-ccdA constructs were transformed in MG1655 expressing the reference ccdBO157 orccdBF genes from the pBAD33 vector. The resulting transformants were plated on LB plates containing chloramphenicol and ampicillin with arabinose (1%). Basal expression of ccdA from the pTac promoter of pKK223-3 in MG1655 is sufficient to test the antitoxicity phenotype. The CcdA variants were considered to be functional when the toxicity of CcdBO157 or CcdBF protein was counteracted, i.e., when strains coexpressing a ccdA variant with the ccdBO157 or ccdBF reference genes were able to grow in the presence of arabinose while strains expressing only the ccdBO157 or ccdBF reference gene were not.

Result We identified a small sequence that presents identity with the 39 region of ccdAO157 and two putative ORFs (Figure 4, A and B). The 2704-bp IR corresponds to that of 1425 bp with the insertion of an IS621 in the sequence presenting identity with ccdAO157.

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