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.