Difference between revisions of "Part:BBa K3114014:Design"

 
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<partinfo>BBa_K3114014 short</partinfo>
 
<partinfo>BBa_K3114014 short</partinfo>
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<partinfo>BBa_K3114014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3114014 SequenceAndFeatures</partinfo>
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===Design Notes===
 
===Design Notes===
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ICARUS was designed to enable purification of large proteins with strong electrostatic potential in their binding pockets using a 6xHis-tag. The part is designed to be attached in-frame with a protein coding region at the C-terminal, which can be accomplished via Golden Gate reaction using MoClo assembly standard overhangs. The predicted structure of the universal spacer, modelled using <i>ab initio</i> and Rosetta comparative modelling creates a "helix-turn-helix-turn-helix" motif. SacII restriction sites are positioned at the start of the spacer and after the 6xHis-tag for removal of the 6x His-tag at the DNA level. In addition, a thrombin proteolytic site exists in the first turn of the predicted motif for 6xHis-tag removal at the protein level. The second turn in the predicted motif is filled with aspartic acid residues to repel electronegative forces, if used with a protein that has a binding pocket that is highly electronegative. This part also includes a double stop codon and a double terminator <a href="https://parts.igem.org/Part:BBa_B0014">(BBa_B0014).</a>  Codons were alternated to avoid tRNA depletion and were optimized based on E. coli molecular machinery.
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===Source===
 
===Source===
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===References===
 
===References===

Revision as of 11:12, 21 October 2019

ICARUS spacer + 6XHis tag + double terminator for purification of highly electronegative proteins


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 215
    Illegal XhoI site found at 318
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 321

Design Notes

ICARUS was designed to enable purification of large proteins with strong electrostatic potential in their binding pockets using a 6xHis-tag. The part is designed to be attached in-frame with a protein coding region at the C-terminal, which can be accomplished via Golden Gate reaction using MoClo assembly standard overhangs. The predicted structure of the universal spacer, modelled using ab initio and Rosetta comparative modelling creates a "helix-turn-helix-turn-helix" motif. SacII restriction sites are positioned at the start of the spacer and after the 6xHis-tag for removal of the 6x His-tag at the DNA level. In addition, a thrombin proteolytic site exists in the first turn of the predicted motif for 6xHis-tag removal at the protein level. The second turn in the predicted motif is filled with aspartic acid residues to repel electronegative forces, if used with a protein that has a binding pocket that is highly electronegative. This part also includes a double stop codon and a double terminator <a href="https://parts.igem.org/Part:BBa_B0014">(BBa_B0014).</a> Codons were alternated to avoid tRNA depletion and were optimized based on E. coli molecular machinery.

Source

wip

References