Cloning of recA gene of Corynebacterium glutamicum and phenotypic complementation of Escherichia coli recombinant deficient strain

摘要

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEMp-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5l recA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5l recA cells revealed tolerance of transformed cells up to dose 0.24 J/cmpo, while non-transformed cells tolerated only up to dose 0.08 J/cmpo. It is concluded that phenotypic complementation of E. coli DH5l recA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.