Part:BBa_K1659211:Design
Dispersin B fused with DsbA, with mutation at nucleotide 636 (T to C)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 316
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 472
Design Notes and Sources
This part is a modified version of BBa_K1659201, where thymine 636 was mutated into a cytosine, conserving the His-212 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_K1659201. 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_K1659201, 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)