Part:BBa_K1659210:Design

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].

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 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)