Vector map of pPconst1326
- E. coli/ Bacillus shuttle vector
- Target gene cloning in E. coli and protein production in Bacillus sp.
- For constitutive expression of recombinant proteins in Bacillus megaterium
- pPconst1326 is an E. coli/ Bacillus shuttle vector for the constitutive expression of recombinant proteins in Bacillus megaterium. pPconst1326 can be used for cloning of a
target gene in E. coli and protein production in Bacillus sp..
- To generate the pPconst1326 constitutive vector the promoter of pyruvate dehydrogenase operon (pdhABCD, bmd_1326-1329) was isolated from
B. megaterium DSM319 and cloned into an E. coli/ Bacillus shuttle vector. The promoter region is followed by the native ribosomal binding site (RBS) of the corresponding gene
cluster including its start codon that is located upstream of multiple cloning site (MCS). First restriction site (SpeI) allows cloning of a gene of interest fused to nine additional
nucleotides encoding Met-Thr-Ser at its N-terminus. The pPconst1326 carries the oriU and repU gene from Bacillus cereus for replication in Bacillus sp., the tetL
gene providing resistance against tetracycline, and functional genetic elements for E. coli (origin of replication ColE1 ori and the bla gene encoding a ß-lactamase
which provides resistance against ampicillin). Unlike inducible gene expression system the expression of the target protein is driven constitutively by the pdhABCD promoter.
Thus, addition of an inducer is not required during the cultivation of B. megaterium cells.