Regulatory
GAL1

Part:BBa_J63006:Experience

Designed by: Caroline Ajo-Franklin   Group: Silver Lab   (2006-10-11)
Revision as of 23:17, 23 September 2011 by Ayjchan (Talk | contribs) (→‎Characterization by British Columbia iGEM 2011)

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UNIQ07d04efe45f4e1fb-partinfo-00000000-QINU UNIQ07d04efe45f4e1fb-partinfo-00000001-QINU

Characterization by British Columbia iGEM 2011

Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters

The GPD promoter (BBa_K517000), GAL promoter (BBa_K517001) and GAL1 promoter with Kozak sequence (BBa_J63006) as characterized by their regulation of the expression of a GFP reporter.

Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters: S. cerevisiae yeast strains containing either the GPD-GFP, GAL-GFP or GAL+Kozak-GFP construct were cultured overnight at 30 degrees Celsius in either YPD (dextrose), SC-raffinose or SC-galactose media. These were diluted 1 in 10 in their respective media and grown for 3 hours at 30 degrees Celsius into log phase. The cells were then spun down and samples were collected at 0 hours. The remaining cells were resuspended in SC-galactose media and left to grow at 30 degrees Celsius for 3 hours. The cells were spun down again and samples were collected at this 3 hour time point. The samples were fixed in paraformaldehyde and visualized under a fluorescence microscope under the GFP and DIC settings. Acquired images were then color-combined with red representing DIC and green representing GFP.

Microscopy images show that the BBa_K517000 GPD promoter is constitutively activated and there is induction of the BBa_K517001 GAL promoter when it is shifted to galactose media. However, it is difficult to differentiate the expression of GFP under the regulation of the BBa_J63006 GAL Promoter with kozak sequence whether in glucose or galactose media. We have also analysed these by fluorescence activated cell sorting (FACS) (data available on BBa_K517000 and BBa_K517001 experience pages) but it remains ambiguous for the BBa_J63006 GAL Promoter. It would appear that were is a very weak constitutive expression of the GFP reporter under the BBa_J63006 GAL Promoter.

DISCLAIMER: It may be that there was an error in cloning. We have to sequence to find out if we placed the BBa_J63006 GAL Promoter accurately into our GFP reporter construct...