Difference between revisions of "Part:BBa K118023:Experience"

Revision as of 22:32, 30 October 2016

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out. Cellulomonas fimi uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose. This part was codon optimized for the expression in Caulobacter crescentus and synthesized by IDT.

We improved the characterization of this part by optimizing the expression and attempted to express the enzyme on the surface layer of C.crescentus. We characterized the activity of the CenA insert in the C.crescentus s-layer. In initial activity analysis we saw no increased activity compared to wild type RsaA protein indicating that either the protein insert as we designed it does not fold correctly in the surface layer, further cloning attempts and design alterations must be made to try to improve the results.

It likely did not work as the folding of the protein may have been disrupted by the insertion into the surface layer; we would have to try different designs of the protein insertion by using different insertion sites or cloning in a altered, truncated, version of the protein.

Applications of BBa_K118023

User Reviews

UNIQ42fe45a95cce9aa1-partinfo-00000000-QINU

BBa_K118023 Allan Crossman (Edinburgh 2011)

Edinburgh originally made this part in 2008. We came back to it in 2011 and found a double "mutation" from the Registry sequence. Upon checking with [http://www.ncbi.nlm.nih.gov/nuccore/NC_015514.1?from=3561504&to=3562853&report=genbank&strand=true a published genome sequence, NC_015514.1], we found that our "mutations" were in fact nothing of the sort, and must have been present all along. So the true sequence of K118023 is almost certainly that published under accession [http://www.ncbi.nlm.nih.gov/nuccore/NC_015514.1?from=3561504&to=3562853&report=genbank&strand=true NC_015514.1] (aside from the stop codons).

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UNIQ42fe45a95cce9aa1-partinfo-00000002-QINU

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 We improved the characterization of this part by optimizing the expression and attempted to express the enzyme on the surface layer of <i>C.crescentus</i>. We characterized the activity of the CenA insert in the <i>C.crescentus</i> s-layer. In initial activity analysis we saw no increased activity compared to wild type RsaA protein indicating that either the protein insert as we designed it does not fold correctly in the surface layer, further cloning attempts and design alterations must be made to try to improve the results.
-It likely didn't work as the folding of the protein may have been disrupted by the insertion into the surface layer, we would have to try different designs of the protein insertion by using different insertion sites or cloning the altering the truncation of the protein.
+It likely did not work as the folding of the protein may have been disrupted by the insertion into the surface layer; we would have to try different designs of the protein insertion by using different insertion sites or cloning in a altered, truncated, version of the protein.
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