Difference between revisions of "Part:BBa K895006"
m (→Contribution) |
Sbelhouari (Talk | contribs) |
||
| Line 13: | Line 13: | ||
Summary: iGEM 2017 Team Tsinghua has made a significant improvement of this part, by validating that this GFP protein works well in the S. cerevisiae (baking yeast) chassis, and adding a yeast inducible promoter pGalS (BBa_K2247000) in front of this part. The new part we constructed is [https://parts.igem.org/Part:BBa_K2247001 BBa_K2247001]. By making this improvement, we achieved inducible expression of GFP by galactose or by yeast Gal4 protein in yeast cells, and this new composite part can also serve to validate and characterize the function of the pGalS promoter. In future projects applying the yeast-two-hybrid or yeast-one-hybrid system, this new part can serve as a basic and standard reporter plasmid. For experimental validation of the new part, please see the main page of BBa_K2247001. | Summary: iGEM 2017 Team Tsinghua has made a significant improvement of this part, by validating that this GFP protein works well in the S. cerevisiae (baking yeast) chassis, and adding a yeast inducible promoter pGalS (BBa_K2247000) in front of this part. The new part we constructed is [https://parts.igem.org/Part:BBa_K2247001 BBa_K2247001]. By making this improvement, we achieved inducible expression of GFP by galactose or by yeast Gal4 protein in yeast cells, and this new composite part can also serve to validate and characterize the function of the pGalS promoter. In future projects applying the yeast-two-hybrid or yeast-one-hybrid system, this new part can serve as a basic and standard reporter plasmid. For experimental validation of the new part, please see the main page of BBa_K2247001. | ||
| + | |||
| + | ==Team uOttawa 2019: Characterization and Improvement of BBa_K895006== | ||
| + | |||
| + | <p><b>Abstract:</b> Team uOttawa creates a translational unit out of <a href="https://parts.igem.org/Part:BBa_K895006">BBa_K895006</a> named <a href="https://parts.igem.org/Part:BBa_K3271028">BBa_K3271028</a> to assess the use of the GFP coding sequence in <i>Saccharomyces cerevisiae</i>. Finding the fluorescence signal to be weak, they improve <a href="https://parts.igem.org/Part:BBa_K895006">BBa_K895006</a> by using a codon sequence optimized for S. cerevisiae and by using a similar translational unit to <a href="https://parts.igem.org/Part:BBa_K3271028">BBa_K3271028</a> (improvement code <a href="https://parts.igem.org/Part:BBa_K3271029">BBa_K3271029</a>) to provide direct comparison between the fluorescence of their proposed GFP translational unit sequence and that of <a href="https://parts.igem.org/Part:BBa_K895006">BBa_K895006</a>. | ||
| + | </p><br> | ||
| + | |||
| + | <p>We created the translational unit BBa_K3271028 (<b>Figure 1</b>) by relying on homologous recombination in <i>S. cerevisiae</i>.<br> | ||
| + | To do so, we made 3 sets of primers which had the following characteristics:<br> | ||
| + | • One set of primers would amplify the GFP open reading frame and create homologous overhangs to the GDP promoter and the CYC1 terminator.<br> | ||
| + | • 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. <br> | ||
| + | • One set of primers would amplify the CYC1 terminator and create a homologous overhang to the GFP open reading frame. | ||
| + | </p><br> | ||
| + | |||
| + | <p>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 (<b>Figure 1</i>). 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. | ||
| + | </p><br> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 00:56, 22 October 2019
GFP open reading frame
This is GFP from Biobrick BBa_J54103, which we have sequenced and re-cloned to RFC[10] standard in pSB1C3.
Contribution
Group: Team Tsinghua 2017
Author: Boyang Gao
Summary: iGEM 2017 Team Tsinghua has made a significant improvement of this part, by validating that this GFP protein works well in the S. cerevisiae (baking yeast) chassis, and adding a yeast inducible promoter pGalS (BBa_K2247000) in front of this part. The new part we constructed is BBa_K2247001. By making this improvement, we achieved inducible expression of GFP by galactose or by yeast Gal4 protein in yeast cells, and this new composite part can also serve to validate and characterize the function of the pGalS promoter. In future projects applying the yeast-two-hybrid or yeast-one-hybrid system, this new part can serve as a basic and standard reporter plasmid. For experimental validation of the new part, please see the main page of BBa_K2247001.
Team uOttawa 2019: Characterization and Improvement of BBa_K895006
Abstract: Team uOttawa creates a translational unit out of <a href="https://parts.igem.org/Part:BBa_K895006">BBa_K895006</a> named <a href="https://parts.igem.org/Part:BBa_K3271028">BBa_K3271028</a> to assess the use of the GFP coding sequence in Saccharomyces cerevisiae. Finding the fluorescence signal to be weak, they improve <a href="https://parts.igem.org/Part:BBa_K895006">BBa_K895006</a> by using a codon sequence optimized for S. cerevisiae and by using a similar translational unit to <a href="https://parts.igem.org/Part:BBa_K3271028">BBa_K3271028</a> (improvement code <a href="https://parts.igem.org/Part:BBa_K3271029">BBa_K3271029</a>) to provide direct comparison between the fluorescence of their proposed GFP translational unit sequence and that of <a href="https://parts.igem.org/Part:BBa_K895006">BBa_K895006</a>.
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</i>). 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.
</p>
Sequence and Features