Part:BBa_K627010
Fusion of AraC induction system and TEV protease 3
Introduction
This BioBrick is a 2 part fusion of arabinose-inducible induction system and the TEV protease.
General Information of the single sub parts
TEV protease
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus (TEV). It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.
At 37°C, the TEV protease forms inclusion body, which leads to an inactive form. Incubated at 30 °C, the protease is expressed as soluble type and is highly active.
Arabinose-inducible induction system
The arabinose-inducible induction system was amplified via PCR from the pBAD_iGEMexpress vector. Based on the inhibitionary funtion of the catabolic activator protein (CAP) it got a very low expression rate without induction by arabinose. For the induction concentration between 2 mM up to 50 mM arabinose can be used to get an high expression rate.
General Function
When arabinose is added to the media (2mM up to 50 mM), the CAP lost its inhibitory function and the arabinose-inducible induction system gets activated. The protein, here the HRV 14_3C protease, after the T7 RBS gets express at a very high rate. So we can assume that this system is an effectiv protease generator.
Performance and Summary
In combination with our created ssTorA_CS-TEV_blaFL device (BBa_K627012, More Information) this protease generator was able to mediate the cell death of our transformed E.coli cells at ampicillin concentration up to 100 µg/ml. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1523
Illegal BglII site found at 1711
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1239
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
This BioBrick is a 2 parts fusion of pBAD arabinose-inducible induction system and the TEV protease.
At 37°C, the TEV protease forms inculsion body, which leads to an inactive form. Incubated at 30 °C, the protease is expressed as soluble type and is highly active. The induction system was amplified via PCR und fused via NgoMIV with the protease.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1239
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Illegal SapI.rc site found at 1563
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