Part:BBa_K627013
Fusion of ssTorA, cleavage site of 14_3C protease and b-lactamase
Introduction
This device is an detector for proteolytic activity of the matching protease (see cleavage site, here for HRV 14_3C 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 Information of the single sub parts
ssTorA
The TorA signal-sequence originate from the enzyme Trimethylamin-N-Oxid-Reduktase, which needs to be folded in the cytoplasm.
beta lactamase
The beta lactamase is an enzyme which cleaves lactam rings and makes bacteria resistant to beta lactam antibiotics like ampicillin and penicillin.
Proteolytic cleavage site for TEV protease
The cleavage site for the HRV 14_3C 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.
Setting up the right order...
This construct contains the cleavage site for the HRV 14_3C protease flanked by the sequence of beta-lactamase at the end and the TorA signal sequence for the TAT (twin arginine transporter) pathway infront of the cleavage site.
General Function
This protease activity detector device provides the signal sequence from TorA for the TAT pathway, called 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, makes this protease activity detector highly adaptable to any desired protease.
Performance and Summary
With co-transformation of the HRV 14_3C 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 117
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 143
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 796
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