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Applications of BBa_J63006
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.
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.
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 20 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, dependence of activation on concentration, related to the 2% concentration.
Characterization by British Columbia iGEM 2011
Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters
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.
Improvement by USTC 2016
Our part is Part: BBa_K2009363 ,designed by Kaiyue Ma. We construted this composite by ligating GAL1 promoter + Kozak sequence (Part:BBa_J63006) and (2) GFP (Part:BBa_E0040). However, the ATG inside the KOZAK sequence won't be in the same reading frame as the ATG of the downstream coding sequence, so a frame-shift mutation is inevitable. To solve this problem, we add a base pair after the Kozak sequence so that the ATG can be in the same reading frame and the GFP can be expressed properly. What’s more, this part become “ready to use”, which means the GFP sequence can be directly altered by other functional parts and the sequence will be expressed properly.
Improvement by iGEM Kyoto 2017
We used this promoter in order to express long hairpin RNA in yeast. Long hairpin RNA targeting B. xylophilus AK1 mRNA or GFP mRNA was cloned downstream of the promoter and introduced into the 2-micron high copy number plasmid of budding yeast, and subsequently expressed in yeast. The GPD promoter (BBa_K517001) was used as a control. For the data, please refer to the GPD Promoter page ( BBa_K571001 ) and our Wiki Result. By feeding yeast expressing dsRNA to nematodes, we investigated whether the dsRNA expressed by these promoters is fatal to pine wood nematodes.