Abstract
In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.
Highlights
DNA dependent RNA polymerase (RNAP) initiates the transcription process in bacteria after binding to a speci¢c DNA sequence named the promoter
The main problem with this strategy is that if a very strong promoter is present in the inserted DNA fragment, the reporter gene product will accumulate in very large amounts inside the cell, resulting in reduced cell viability
We have constructed a vector based on the ampicillin resistance gene of the pBR322 plasmid whose product is secreted into the periplasmic space of E. coli
Summary
DNA dependent RNA polymerase (RNAP) initiates the transcription process in bacteria after binding to a speci¢c DNA sequence named the promoter. The main problem with this strategy is that if a very strong promoter is present in the inserted DNA fragment, the reporter gene product will accumulate in very large amounts inside the cell, resulting in reduced cell viability. To avoid this problem, we have constructed a vector based on the ampicillin resistance gene of the pBR322 plasmid whose product is secreted into the periplasmic space of E. coli. DNA was sequenced on both strands using the fmol DNA Sequencing kit (Promega) [24] after puri¢cation with the Wizard Clean Up system (Promega)
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call