Difference between revisions of "Part:BBa K627012"

Revision as of 22:41, 21 September 2011

Fusion of TorA sig-seq, TEV protease cleavage site and b-lactamase

This construct contains the cleavage site for the TEV protease flanked by the sequence of beta-lactamase and the TorA signal sequence for the TAT (twin arginine transporter) pathway, thus in case of the expression of the complete construct the beta lactamase can be transported into periplasm and provide ampicillin resistance. The TorA signal-sequence originate from the enzyme Trimethylamin-N-Oxid-Reduktase , which needs to be folded in the cytoplasm. The beta lactamase is an enzyme which cleaves lactam rings and makes bacteria resistant to antibiotics like ampicillin and penicillin. The cleavage site for the TEV protease was created via oligo hybridization and ligated into the construct with cleavage site for XhoI and NheI, which makes this part modular and easy adaptable for any other protease.

Background

This device is an detector for proteolytic activity of the matching protease (see cleavage site, here for TEV protease) inside the cytoplasm of E.coli. If there is an active protease, than the resistance capacity against ampicillin is lowered and the cells won't survive on higher antibiotic concentrations.

General Function

This protease activity detector device provides an export sequence for the TAT pathway, the ssTorA. These signal sequence is used for the export of folded proteins into the periplasm of gram-negative bacteria like E.coli.

The modular cleavage site, which can be exchanged with the enzymes XhoI and NheI, can be adapted to any protease. This feature allows the user to check for specific proteolytic activity in the cytoplasm.

The beta lactamase, a popular resistance marker, is used to check the activity of the desired protease inside the cytoplasm. By using the TAT pathway to export the lactamase into the periplasm the survival of bacteria cells at high ampicillin concentrations shows a low protease activity. In contrast, the death of all colony forming units indicates a high proteolytic activity inside the cytoplasm, which leads to a reduced export capacity of beta lactamase via the TAT pathway.

Also the modular construction of the cleavage site, which can be exchanged with the restriction enzymes XhoI and NheI, this protease activity detector is highly adaptable to any desired protease.

Performance and Summary

With co-transformation of the TEV protease on a second plasmid, we can show the desired proof of principal: when the protease expression was activated, the transformed E.coli cells did not grow at ampicillin concentrations up to 100 µg/ml. The control colonies without induction of the protease survived up to concentrations of 400 µg/ml ampicillin.

Sequence and Features