Regulatory

Part:BBa_K165001

Designed by: John Szymanski   Group: iGEM08_BrownTwo   (2008-10-26)
Revision as of 03:58, 31 October 2008 by Jszymans (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

pGAL1+ w/XhoI sites

This is the GAL1 inducible promoter for use in Saccharomyces cerevisiae. It 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


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


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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 469
    Illegal XhoI site found at 477
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 431
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//rnap/eukaryote/yeast
//direction/forward
//chassis/eukaryote/yeast
//promoter
//regulation/positive
Parameters
negative_regulators
positive_regulators1