Part:BBa_K3271028
pGPD-eGFP-tCYC1 in pSB1K3
To asses the expression of eGFP (BBa_K895006), it was put under the control of constitutive GPD promoter and CYC1 terminator. It was then targeted to S. cerevisiae's Ura3 locus via homologous recombination. Weak fluorescence signal was detected via fluorescence microscopy. This GFP can be used to measure promoter strength or in combination with another protein to determine succesful expression. This BioBrick part can also be transformed within BioBrick backbones BBa_K3271004, BBa_K3271005, BBa_K3271006, BBa_K3271007, BBa_K3271012, BBa_K3271014, BBa_K3271015, BBa_K3271016, BBa_K3271021, BBa_K3271022, BBa_K3271023, and BBa_K3271024, to learn cloning in S. cerevisiae via homologous recombination. Please the Registry pages of each respective BioBrick backbones for additional details.
Team uOttawa 2019: Characterization and Improvement of BBa_K895006
Abstract: Team uOttawa creates a translational unit out of BBa_K895006 named BBa_K3271028 to assess the use of the GFP coding sequence in Saccharomyces cerevisiae. Finding the fluorescence signal to be weak, they improve BBa_K895006 by using a codon sequence optimized for S. cerevisiae and by using a similar translational unit to BBa_K3271028 (improvement code BBa_K3271029 to provide direct comparison between the fluorescence of their proposed GFP translational unit sequence and that of BBa_K895006.
We created the translational unit BBa_K3271028 (Figure 1) by relying on homologous recombination in S. cerevisiae.
To do so, we made 3 sets of primers which had the following characteristics:
• One set of primers would amplify the GFP open reading frame and create homologous overhangs to the GDP promoter and the CYC1 terminator.
• One set of primers would amplify from the URA 3 promoter to the GDP promoter and create a homologous overhang to the GFP open reading frame.
• One set of primers would amplify the CYC1 terminator and create a homologous overhang to the GFP open reading frame.
After performing the 3 PCRs, we had 3 different DNA parts with homologous regions of overhang. We then did a yeast transformation and relied on the yeast to perform homologous recombination to fuse the homologous regions together (Figure 1). We used the BY4742 strain of yeast because it has a URA3 gene mutation that makes URA3 dysfunctional; this technically means that if the yeast incorporated our DNA, it would would grow on Ura- selection.
Following the yeast transformation we did a colony PCR to verify that our transformation worked. Once confirmed, we moved on to using a confocal microscope and ImageJ software to characterize the part. We compared the intensity of the GFP open reading frame from the construct to that of the yeast strain BY4742 as the negative control. It is evident that there is basically no fluorescence from the yeast strain BY4742 (as there shouldn't be).
First, using the confocal microscope, we randomly chose 11 different sampling areas at the same magnification from the slide; we then took both Brightfield and fluorescent images of these sampling areas. Of the 11 sets of sample images, we chose 7 to analyze. To analyze the images, we opened the fluorescent image from each set in ImageJ. We began by using the Brightfield image (pulled up on another screen) to help us select an area on the fluorescent image where there were no cells. Then, we recorded the area, min & max gray value, integrated density, and mean gray value of the selected area. Next, we used the process tool to subtract the mean gray value of the selected area. Once this was done, we measured the min & max gray value, integrated density, and mean gray value of the entire image. Separately, we counted the amount of cells in each sample using the Brightfield image; note that a budding cell was counted as a single cell. Finally, we divided the integrated density by the number of cells in a given sample to find the fluorescence of each cell. Below is the data obtained:
Sample #3:
Sample #6:
Sample #7:
Sample #8:
Sample #9:
Sample #10:
Sample #11:
Note that the average fluorescence of each cell is 19331.989 + 21560.915 + 22030.827 + 24530. 030 + 20018.583 + 28719.409 + 28534.513 = 164726.266/7=23532.324 RFU
Negative (Strain BY4742):
Thus, the fluorescence of BBa_K895006 in our translational unit as compared to the wild-type yeast strain looks like:
Using a similar experimental strategy we built an improved translational unit, BBa_K3271029, which is identical to the one depicted in Figure 1, except that the GFP from BBa_K895006 is replaced by GFP in which the codons were optimized for expression in yeast (yeGFP).
This improved translational unit visibly fluoresced more as can be seen by the microscopy pictures below. The improved translational unit was also assessed by fluorescence microscopy in the strain W303a.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1179
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