Difference between revisions of "Part:BBa K627009"
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<partinfo>BBa_K627009 short</partinfo>
 
<partinfo>BBa_K627009 short</partinfo>
  
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This BioBrick is a 2 parts fusion of pBAD arabinose-inducible induction system and the 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). TEV protease is a useful reagent for cleaving fusion proteins. 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.
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====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.<br>
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<br>
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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.
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=====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 TEV 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, [https://parts.igem.org/Part:BBa_K627012 More Information]) this protease generator was able to mediate the cell death of our transformed <i>E.coli</i> cells at ampicillin concentration up to 100 µg/ml.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K627011 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K627011 parameters</partinfo>
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<!-- -->
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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.
 
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.
  

Revision as of 23:29, 21 September 2011

Arabinose inducible TEV protease

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 TEV 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1523
    Illegal BglII site found at 1711
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1239
  • 1000
    INCOMPATIBLE 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1239
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 1563