Hi, Does anyone know pet28 cloning vecot can express in E.coli? I have to clone two genes in to pseudomonas and I dont know whether pet28 plasmid derived from E.coli can whould be successful. On the other hand, Pet28 plasmid can replicate in pseudomonas?
pET28 will NOT replicate in Pseudomonas. It will replicate in all strains of E. coli but it will only express in certain strains of E. coli that carry the T7 polymerase gene. These usually carry the designation DE3 (although there are some exceptions to this).
If I remember correctly, vectors with the pBR322/pMB1/ColE1/pUC* origin of replication (different names for the same plasmid oris) don't replicate in Pseudomonas species because a critical RNA for plasmid replication is not expressed. The Pseudomonas DNA-directed RNA polymerase simply does not recognize the sequence upstream of it as a promoter. The vast majority of vectors out there use replication origins derived from E. coli which either function poorly or not at all outside of the Enterobacterales taxonomic order (which includes E. coli).
Most vectors designed to replicate in Pseudomonas utilize unusual origins of replication, derived from natural broad host range plasmids, which function in many organisms distantly related to each other. These plasmid origins conveniently also replicate in E. coli.
*Technically the pUC sequence has a few mutations to the origin that increase its copy number compared to pBR322/pMB1/ColE1 but it's essentially the same otherwise.
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