Part:BBa_J63006:Experience

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.

User Reviews

UNIQ53c6f2b82349facd-partinfo-00000000-QINU

••• British Columbia iGEM 2011

We have not yet sequenced but only used PCR to determine accurate placement of promoter and kozak sequence in front of GFP reporter. Please refer to characterization data below for more details. However, based on our results and analysis, we recommend using our BBa_K517000 GAL promoter, which may be easier to connect to your construct since it lacks the ATG-containing Kozak Sequence present in the BBa_J63006 GAL promoter. Again, please refer to our characterization for more details. •••• Team:Shenzhen 2012

We have re-engineered this part to a new one BBa_K809605. We mutate the ATG at the 5' end to ACG, so it would not work as start codon. This could be useful for fusion protein construction and other purpose.

UNIQ53c6f2b82349facd-partinfo-00000005-QINU

Characterization by British Columbia iGEM 2011

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

The GPD promoter (BBa_K517001), GAL promoter (BBa_K517000) 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_K517001 GPD promoter is constitutively activated and there is induction of the BBa_K517000 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.

As a follow up to this experiment, we did a quick check of the BBa_J63006 GAL Promoter, but results were again not conclusive; there does not seem to be a clear induction of GFP expression when grown in galactose media.

Fluorescence Analysis of GFP expression as regulated by the GAL Promoters: S. cerevisiae yeast strains containing the GAL+Kozak-GFP construct were cultured at 30 degrees Celsius in either YPD (glucose) or SC-galactose media for 3 hours into log phase. The cells were 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.

DISCLAIMER: It may be that there was some error in cloning. We have to sequence to find out if we placed the BBa_J63006 GAL Promoter accurately into our GFP reporter construct. Nonetheless, PCR has been performed to confirm the presence of the BBa_J63006 GAL Promoter upstream of the GFP reporter. We are currently sequencing this construct and will have details at the Regional Jamboree 2011.

Another potential explanation for why this part may not have worked compared to our own BBa_K517000 GAL Promoter is that the kozak sequence may be interfering with expression of protein downstream; since the kozak sequence itself carries an ATG, it may be possible that this is competing with the ATG of the GFP to result in less expression even under galactose induction.

We aligned this (1) BBa_J63006 GAL Promoter+Kozak Sequence against our (2) BBa_K517000 GAL Promoter. There is 180bp upstream sequence present in (1) that is absent in (2), as well as the 27bp Kozak Sequence downstream of the identical sequence regions.