Difference between revisions of "Part:BBa J63006:Experience"

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[[Image:Ubcigem2011Promoterimages.jpg | frame | center | '''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. ]]
 
[[Image:Ubcigem2011Promoterimages.jpg | frame | center | '''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. ]]
  
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Microscopy images show that the 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.
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Microscopy images show that the <partinfo>BBa_K517000</partinfo> GPD promoter is constitutively activated and there is induction of the <partinfo>BBa_K517001</partinfo> GAL promoter when it is shifted to galactose media. However, it is difficult to differentiate the expression of GFP under the regulation of the <partinfo>BBa_J63006</partinfo> 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 <partinfo>BBa_K517000</partinfo> and <partinfo>BBa_K517001</partinfo> experience pages) but it remains ambiguous for the <partinfo>BBa_J63006</partinfo> GAL Promoter. It would appear that were is a very weak constitutive expression of the GFP reporter under the <partinfo>BBa_J63006</partinfo> GAL Promoter.
  
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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...
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DISCLAIMER: It may be that there was an error in cloning. We have to sequence to find out if we placed the <partinfo>BBa_J63006</partinfo> GAL Promoter accurately into our GFP reporter construct...
  
 
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Revision as of 23:17, 23 September 2011

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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...