Difference between revisions of "Part:BBa K165046"

Revision as of 03:57, 31 October 2008

pGAL1+ Kozak

The pGAL promoter is induced by the presence of galactose and repressed by glucose. Raffinose is used as a neutral carbon source, with no effect on pGAL1. The promoter normally has an all-or-nothing response. To make it tunable, knock out the Gal2 gene.[http://www.jbc.org/cgi/content/abstract/M512317200v1]

Use these primers on a Kan resistance gene template: (the portion in bold binds to the template, the rest is homologous to the Gal2 gene on the yeast chromosome.)

GAL2ko.fwd: AGCAGCAAAA CATTAATTTT GCTTCCAAGA CGACAGTAAT ATGTCTCCTA CAATACCAGT GGCAGATCCGCTAGGGATAA
GAL2ko.rev: AATACTCTGATATATGTACACAAATAATAGGTTTAGGTAAGGAATTTATATAATCGTAAG TTCGAGCTCGTTTAAAC

To check for proper knockout, amplify the section of the chromosome where the insert should be. Use these primers:

GAL2kosc.fwd: TGTGCATGTT ATCTATATCC TTCTTTATAT AGATGCTGTT
GAL2kosc.rev: ATT AAT TGT ATG TTA GCT CAG GAA TTC AAC TGG AAG AAA G

Thanks to the lab of Christina Smolke for these primer sequences.

Transform the product into yeast by standard procedure [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php], and select transformants with G418 YPD plates

Sequence and Features