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<I>British Columbia iGEM 2011</I>
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Inconclusive results on our part since we did not sequence to confirm accurate placement of promoter and kozak sequence in front of GFP reporter. Please refer to characterization data below for more details.
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Revision as of 23:19, 23 September 2011

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.

Applications of BBa_J63006

User Reviews

UNIQa5dc18f8855c34b5-partinfo-00000000-QINU

•••

British Columbia iGEM 2011

Inconclusive results on our part since we did not sequence to confirm accurate placement of promoter and kozak sequence in front of GFP reporter. Please refer to characterization data below for more details.

UNIQa5dc18f8855c34b5-partinfo-00000002-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...