Difference between revisions of "Part:BBa K1659210:Design"
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This part is a modified version of [https://parts.igem.org/Part:BBa_K1659200:Design BBa_K1659200], where thymine 582 was mutated into a cytosine, conserving the His-194 which it encodes for. The site-directed mutagenesis was performed using a modified [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis NEB Q5 Mutagenesis Protocol], which is fully described in our team's [http://2015.igem.org/Team:Oxford/Protocols Protocols Page].  
 
This part is a modified version of [https://parts.igem.org/Part:BBa_K1659200:Design BBa_K1659200], where thymine 582 was mutated into a cytosine, conserving the His-194 which it encodes for. The site-directed mutagenesis was performed using a modified [https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis NEB Q5 Mutagenesis Protocol], which is fully described in our team's [http://2015.igem.org/Team:Oxford/Protocols Protocols Page].  
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Image: The base sequence shown is the offending section of BBa_K1659200, and the overlapping primers for the mutagenesis are overlaid. The primers were designed using [https://www.snapgene.com/ SnapGene] [1].  
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Image: The base sequence shown is the offending section of BBa_K1659200, and the overlapping primers for the mutagenesis are overlaid. The primers were designed using <a href="http://www.snapgene.com/">SnapGene</a> [1].  
 
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Revision as of 17:11, 13 September 2015

Dispersin B with mutation at nucleotide 582 (T to C)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 262
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 418


Design Notes and Sources

This part is a modified version of BBa_K1659200, where thymine 582 was mutated into a cytosine, conserving the His-194 which it encodes for. The site-directed mutagenesis was performed using a modified NEB Q5 Mutagenesis Protocol, which is fully described in our team's [http://2015.igem.org/Team:Oxford/Protocols Protocols Page].



DspB quikchange

The mutation created was necessary for the removal of the BspHI restriction site found within the coding sequence of BBa_K1659200. This is to help facilitate the characterization of the part, as we characterize our coding sequence parts by cloning them into the commercial expression vector [https://www.thermofisher.com/order/catalog/product/V43001 pBAD/HisB], the process of which requires restriction digest at the BspHI site upstream of the part added on via PCR for ligation with the NcoI site found on the vector.


Image: The base sequence shown is the offending section of BBa_K1659200, and the overlapping primers for the mutagenesis are overlaid. The primers were designed using SnapGene [1].




References

[1] SnapGene software (from GSL Biotech; available at snapgene.com)