Difference between revisions of "Part:BBa K1744000:Design"

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<partinfo>BBa_K1744000 short</partinfo>
 
<partinfo>BBa_K1744000 short</partinfo>
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<partinfo>BBa_K1744000 SequenceAndFeatures</partinfo>
 
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===Design Notes===
 
===Design Notes===
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The protein produced when the expression of vcrx028 is induced is a toxin, so you MUST repress this arabinose killswitch by adding glucose. Otherwise, you will get either no positive clones or highly mutated toxin that won't work well. In fact, the toxin sequence used is mutated from the native one, but proven good killing efficiency when induced. The part could not be BioBrick standardized for XbaI and PstI. It can still be digested using NotI or EcoRI + SpeI.
 
The protein produced when the expression of vcrx028 is induced is a toxin, so you MUST repress this arabinose killswitch by adding glucose. Otherwise, you will get either no positive clones or highly mutated toxin that won't work well. In fact, the toxin sequence used is mutated from the native one, but proven good killing efficiency when induced. The part could not be BioBrick standardized for XbaI and PstI. It can still be digested using NotI or EcoRI + SpeI.
 +
  
 
===Sources===
 
===Sources===
  
 
A RBS was selected through rational design and added with a primer to pVCR94's toxin coding sequence and it was cloned in the plasmid pBAD30 (using the site EcoRI). The part itself is a region of the resulting plasmid, from araC to bla.
 
A RBS was selected through rational design and added with a primer to pVCR94's toxin coding sequence and it was cloned in the plasmid pBAD30 (using the site EcoRI). The part itself is a region of the resulting plasmid, from araC to bla.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1744000 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K1744000 parameters</partinfo>
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Revision as of 07:05, 15 September 2015

araC-PBAD-vcrx028-ampR

Design Notes

The protein produced when the expression of vcrx028 is induced is a toxin, so you MUST repress this arabinose killswitch by adding glucose. Otherwise, you will get either no positive clones or highly mutated toxin that won't work well. In fact, the toxin sequence used is mutated from the native one, but proven good killing efficiency when induced. The part could not be BioBrick standardized for XbaI and PstI. It can still be digested using NotI or EcoRI + SpeI.


Sources

A RBS was selected through rational design and added with a primer to pVCR94's toxin coding sequence and it was cloned in the plasmid pBAD30 (using the site EcoRI). The part itself is a region of the resulting plasmid, from araC to bla.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1619
    Illegal PstI site found at 1631
    Illegal PstI site found at 2708
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1227
    Illegal PstI site found at 1631
    Illegal PstI site found at 2708
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1166
    Illegal BamHI site found at 1613
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1619
    Illegal PstI site found at 1631
    Illegal PstI site found at 2708
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1619
    Illegal PstI site found at 1631
    Illegal PstI site found at 2708
    Illegal AgeI site found at 1001
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1815
    Illegal BsaI site found at 2882
    Illegal SapI site found at 983