Difference between revisions of "Part:BBa K1659210:Design"
Weikongquee (Talk | contribs) |
Weikongquee (Talk | contribs) |
||
| Line 15: | Line 15: | ||
<img src="https://static.igem.org/mediawiki/parts/c/c8/Oxford15_DspBQC1.png" alt="DspB quikchange" width="345" height="275" style="float:right"> | <img src="https://static.igem.org/mediawiki/parts/c/c8/Oxford15_DspBQC1.png" alt="DspB quikchange" width="345" height="275" style="float:right"> | ||
<figcaption> | <figcaption> | ||
| − | <p style="font-size: | + | <p style="font-size:13px"> |
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 <a href="http://www.thermofisher.com/order/catalog/product/V43001">pBAD/HisB</a>, 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. | 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 <a href="http://www.thermofisher.com/order/catalog/product/V43001">pBAD/HisB</a>, 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. | ||
<br> | <br> | ||
Revision as of 17:12, 13 September 2015
Dispersin B with mutation at nucleotide 582 (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 262
- 1000INCOMPATIBLE 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)