Team:The University of Alberta/Butanerd

{| align="center" [ http://2008.igem.org/wiki/images/a/af/HOMEUAB.png] [ http://2008.igem.org/wiki/images/7/7c/TEAMUA.png] [ http://2008.igem.org/wiki/images/7/7e/PROJECTUA.png] [ http://2008.igem.org/wiki/images/c/c0/PARTSUA.png] [ http://2008.igem.org/wiki/images/f/f9/NOTEBOOKUA.png] The Butanerd Project Continues!

For background information on the butanol project, please view the iGEM 2007: Alberta team page. Inserts and Promoters/Vectors Used

The following are the five butanol inserts we have been working with this year:
 * 1) RBS + B-hydroxy butyryl CoA dehydrogenase (AKA. 21)
 * 2) RBS + Enoyl-CoA hydratase (AKA. 22)
 * 3) RBS + Butyryl CoA dehydrogenase (AKA. 23)
 * 4) RBS + Butyraldehyde dehydrogenase (AKA. 24)
 * 5) RBS + Butanol dehydrogenase (AKA. 25)
 * All the parts submitted by last year's team can be found here.

The promoters and vector used are:
 * J61003 (without GFP)
 * I0500
 * pSB1A3

The Summer Summary


 * The summer began with working on the butanol project as a training exercise to get the team familiarized with lab procedures. The team worked on ligating individual butanol inserts into the J61003 and I0500 vectors. Furthermore, protein expressions were to be done on these plasmids once the ligations were successfully completed. The ligations were a success by the beginning of July.


 * The team proceeded to show protein expression of the individual butanol inserts. The Western blots were successfully completed by the end of July.


 * Protein Expression of Individual Butanol Inserts!


 * [[image:Individual Butanol Protein Expression.jpg|300px]]
 * Lanes:
 * RBS + B-hydroxy butyryl CoA dehydrogenase -- 33kD
 * RBS + Enoyl-CoA hydratase -- 31kD
 * RBS + Butyryl CoA dehydrogenase -- 44kD
 * RBS + Butyraldehyde dehydrogenase -- 96.8kD
 * RBS + Butanol dehydrogenase -- 45kD


 * Finally, with the eagerness of the team to complete last year's project, the completion of the full butanol operon was adopted as a side project. Last year's intended operon (from insert 21 to insert 25) was successfully assembled in PSB1A3 by mid-October!


 * The Five Insert Butanol Operon!


 * [[image:Five Insert Butanol operon.jpg|300px]]
 * Lanes:


 * Ladder
 * Uncut Plasmid
 * Digestion of the operon with EcoRI and PstI. The butanol operon is expected to be ~6.5kb and the pSB1A3 plasmid is expected to be ~2.2kb.


 * In conclusion, last year's intended operon included the butanol inserts:
 * RBS + B-hydroxy butyryl CoA dehydrogenase
 * RBS + Enoyl-CoA hydratase
 * RBS + Butyryl CoA dehydrogenase
 * RBS + Butyraldehyde dehydrogenase
 * RBS + Butanol dehydrogenase
 * However, the full optimal butanol operon requires three addition inserts:
 * ETFA (electron-transfer-flavoprotein, alpha polypeptide)
 * ETFB (electron-transfer-flavoprotein, beta polypeptide)
 * Thiolase
 * for butanol expression. It is expected that ETFA and ETFB are required along with the five original butanol inserts to produce butanol. Thiolase will help overexpress the production of butanol. Therefore, there is still work required to be done on the our butanol operon.

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