Cloning of rpoE10 and its deletion derivatives in a low copy, broad host range expression vector, pMMB206:
To examine the effect of expression of a wild copy of the rpoE10gene in rpoE10:: km mutant, a wild-type copy of the rpoE10gene was supplied to the rpoE10:: km by cloning the entire coding region of rpoE10 in an expression vector. The gene encoding RpoE10 was amplified by PCR using DreamTaq DNA polymerase (Fermentas), primer pairs RpoE10 -F’ and RpoE10- R’ having PstIand HindIII restriction overhangs in their 5’ends, respectively. The gel-purified PCR product was digested using PstI andHindIII , purified again, and ligated with compatible ends downstream of IPTG inducible lacUV 5 promoter region in a broad host range expression vector, pMMB206. The resulting plasmid (pAPD7) was conjugatively mobilized into A. brasilense , and exconjugants selected on plates containing chloramphenicol. The deletion derivatives RpoE10(Del1) (pAPD8) and RpoE10(Del2) (pAPD9) were constructed as described earlier 19.