Difference between revisions of "Part:BBa J63006:Experience"
(→Characterization by Czech Republic iGEM 2015) |
(→Characterization by Czech Republic iGEM 2015) |
||
| Line 38: | Line 38: | ||
The activity of pGAL1 promoter was characterized by transforming pGAL1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time. | The activity of pGAL1 promoter was characterized by transforming pGAL1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time. | ||
| − | [[File:J63006Timeresponse.png| | + | [[File:J63006Timeresponse.png|500px|left|border]] |
| − | [[File:J63006Timeactivity.png| | + | [[File:J63006Timeactivity.png|500px|left]] |
3 different concentrations (0.5%, 1%, 2%) of galactose were added to 100 µl yeast culture in SD min medium. We measured the fluorescence in 60 minutes intervals for 6 hours using a plate reader. | 3 different concentrations (0.5%, 1%, 2%) of galactose were added to 100 µl yeast culture in SD min medium. We measured the fluorescence in 60 minutes intervals for 6 hours using a plate reader. | ||
| Line 53: | Line 53: | ||
| − | [[File:J63006_activity.png| | + | [[File:J63006_activity.png|392px|left]] |
<html><div style="clear: both;"></div></html> | <html><div style="clear: both;"></div></html> | ||
Revision as of 19:48, 18 September 2015
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
UNIQ4f409c41cfb1e558-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. |
UNIQ4f409c41cfb1e558-partinfo-00000005-QINU
Characterization by Czech Republic iGEM 2015
The activity of pGAL1 promoter was characterized by transforming pGAL1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time.
3 different concentrations (0.5%, 1%, 2%) of galactose were added to 100 µl yeast culture in SD min medium. We measured the fluorescence in 60 minutes intervals for 6 hours using a plate reader.
The first graph shows evolution of fluorescence in time. We subtracted fluorescence of negative control from the fluorescence of each sample and divided this by difference between sample´s and negative control´s ODs in order to obtain RFU units.
Our measurement showed that the higher concentrations causes quicker and higher activation of the promoter.
Since 2% appears to be the convenient concentration for maximal activation of the promoter in our experiment we used it as a reference and related the activity of other concentration to this concentration. Results are displayed in the second graph.
The third graph show the static response characteristic related to the 2% concentration.
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.
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.
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.
