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.

Figure 1: Survival test at different ampicillin concentrations: A) approx. 1000 cfu platet on agar plates containing chloramphenicol; B)Influence of resistance induction with 1 mM IPTG, approx. 1000 cfu where platet on agar plates containing chloramphenicol and 1mM IPTG; C)Reduced amount of E.coli colonies, approx. 1000 cfu were platet on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/ml ampicillin; D)Reduced amount of E.coli colonies, approx. 1000 cfu were platet on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/ml ampicillin;E)Reduced amount of E.coli colonies, approx. 1000 cfu were platet on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/ml ampicillin; F)Reduced amount of E.coli colonies, approx. 1000 cfu were platet on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/ml ampicillin; G)No culture growth of E.coli colonies, approx. 1000 cfu were platet on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/ml ampicillin

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 117
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 143
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 796