Difference between revisions of "Part:BBa K5351001"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K5351001 short</partinfo> | <partinfo>BBa_K5351001 short</partinfo> | ||
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− | <!-- Add more about the biology of this part here | + | <!-- Add more about the biology of this part here --> |
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K5351001 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5351001 SequenceAndFeatures</partinfo> | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K5351001 parameters</partinfo> | <partinfo>BBa_K5351001 parameters</partinfo> | ||
− | < | + | --> |
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+ | __TOC__ | ||
+ | |||
+ | <span id="origin"></span> | ||
+ | = Origin = | ||
+ | |||
+ | Synthesized by the team and constructed as a gRNA plasmid for yeast. | ||
+ | |||
+ | <span id="properties"></span> | ||
+ | = Properties = | ||
+ | |||
+ | This part is designed to mutate the NFS1 gene in yeast, enhancing the metabolism of xylose, an essential step for efficient ethanol production. | ||
+ | |||
+ | <span id="usage-and-biology"></span> | ||
+ | = Usage and Biology = | ||
+ | |||
+ | pSCm-NFS1mu is a gRNA plasmid designed to induce a mutation in the NFS1 gene in yeast. The mutation promotes xylose metabolism, making this part significant for biofuel production. The design of the plasmid includes the SNR52 promoter and SUP4 terminator, which regulate the expression of gRNA targeting the NFS1 gene. | ||
+ | |||
+ | <span id="experimental-approach"></span> | ||
+ | = Experimental Approach = | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/1.png" width="50%" style="display:block; margin:auto;" alt="Plasmid map of pSCm-NFS1" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 1. Plasmid map of pSCm-NFS1.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | The pSCm-N20 plasmid was digested using BsaI, generating fragments of 5984 bp, 441 bp, and 571 bp. The 5984 bp fragment was used as the backbone for constructing the gRNA plasmid. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/2.jpg" width="50%" style="display:block; margin:auto;" alt="Gel electrophoresis of pSCm-N20" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 2. Gel electrophoresis of pSCm-N20.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | The N20 oligos gRNA-492I-F and gRNA-492I-R were synthesized and annealed to form a double-stranded sequence. The gRNA backbone was ligated to the fragments, and the resulting plasmid was transformed into *E. coli* DH5α. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/3.png" width="50%" style="display:block; margin:auto;" alt="Transformation plate of pSCm-NFS1" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 3. Transformation plate of pSCm-NFS1.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | Colonies were verified through colony PCR, and the correct fragment length of 288 bp was obtained. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/4.jpg" width="50%" style="display:block; margin:auto;" alt="Gel electrophoresis validation of pSCm-NFS1" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 4. Gel electrophoresis validation of pSCm-NFS1.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | Sequencing confirmed the successful construction of pSCm-NFS1, showing the correct sequence. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/5.png" width="50%" style="display:block; margin:auto;" alt="Sequencing map of pSCm-NFS1" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 5. Sequencing map of pSCm-NFS1.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | The pSCm-NFS1 plasmid was introduced into yeast strains to induce mutations, leading to enhanced xylose metabolism. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/6.png" width="50%" style="display:block; margin:auto;" alt="PCR verification of mutated strains" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 6. PCR verification of mutated strains.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | Xylose metabolism capabilities were evaluated through a solid medium assay. Mutated strains demonstrated significantly improved xylose utilization compared to wild-type strains. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/7.jpg" width="50%" style="display:block; margin:auto;" alt="Xylose metabolism plate assay" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 7. Xylose metabolism plate assay.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | Fermentation experiments were conducted to measure ethanol production. High-performance liquid chromatography (HPLC) showed that the NFS1 mutant strain produced significantly more ethanol compared to the wild-type strain. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/8.jpg" width="50%" style="display:block; margin:auto;" alt="HPLC results of ethanol production" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 8. HPLC results of ethanol production.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/9.jpg" width="50%" style="display:block; margin:auto;" alt="Colony PCR verification of xylose metabolism strains" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 9. Colony PCR verification of xylose metabolism strains.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | The xylose utilization and ethanol production of the engineered strains were further validated through fermentation assays. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/10.jpg" width="50%" style="display:block; margin:auto;" alt="Growth and ethanol production of xylose metabolism strains" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 10. Growth and ethanol production of xylose metabolism strains.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | The results confirm the success of genetic modifications to enhance xylose metabolism in yeast. | ||
+ | |||
+ | <html> | ||
+ | <div style="text-align:center;"> | ||
+ | <img src="https://static.igem.wiki/teams/5351/bba-k5351001/11.png" width="50%" style="display:block; margin:auto;" alt="Comparison of xylose metabolism and ethanol production" > | ||
+ | <div style="text-align:center;"> | ||
+ | <caption>Figure 11. Comparison of xylose metabolism and ethanol production.</caption> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
+ | <span id="reference"></span> | ||
+ | = Reference = | ||
+ | |||
+ | Wei F., Li M., Wang M., et al. (2020). A C6/C5 co‐fermenting *Saccharomyces cerevisiae* strain with the alleviation of antagonism between xylose utilization and robustness. *GCB Bioenergy*, 13(1), 83–97. | ||
+ | |||
+ | Brat D., Boles E., Wiedemann B. (2009). Functional expression of a bacterial xylose isomerase in *Saccharomyces cerevisiae*. *Applied Microbiology and Biotechnology*, 75, 2304-2311. |
Revision as of 09:25, 27 September 2024
pSCm-NFS1mu
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1135
Illegal NheI site found at 4384
Illegal NotI site found at 2401 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 7707
Illegal BglII site found at 7904
Illegal BamHI site found at 2246
Illegal BamHI site found at 2357
Illegal XhoI site found at 2408 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Origin
Synthesized by the team and constructed as a gRNA plasmid for yeast.
Properties
This part is designed to mutate the NFS1 gene in yeast, enhancing the metabolism of xylose, an essential step for efficient ethanol production.
Usage and Biology
pSCm-NFS1mu is a gRNA plasmid designed to induce a mutation in the NFS1 gene in yeast. The mutation promotes xylose metabolism, making this part significant for biofuel production. The design of the plasmid includes the SNR52 promoter and SUP4 terminator, which regulate the expression of gRNA targeting the NFS1 gene.
Experimental Approach
The pSCm-N20 plasmid was digested using BsaI, generating fragments of 5984 bp, 441 bp, and 571 bp. The 5984 bp fragment was used as the backbone for constructing the gRNA plasmid.
The N20 oligos gRNA-492I-F and gRNA-492I-R were synthesized and annealed to form a double-stranded sequence. The gRNA backbone was ligated to the fragments, and the resulting plasmid was transformed into *E. coli* DH5α.
Colonies were verified through colony PCR, and the correct fragment length of 288 bp was obtained.
Sequencing confirmed the successful construction of pSCm-NFS1, showing the correct sequence.
The pSCm-NFS1 plasmid was introduced into yeast strains to induce mutations, leading to enhanced xylose metabolism.
Xylose metabolism capabilities were evaluated through a solid medium assay. Mutated strains demonstrated significantly improved xylose utilization compared to wild-type strains.
Fermentation experiments were conducted to measure ethanol production. High-performance liquid chromatography (HPLC) showed that the NFS1 mutant strain produced significantly more ethanol compared to the wild-type strain.
The xylose utilization and ethanol production of the engineered strains were further validated through fermentation assays.
The results confirm the success of genetic modifications to enhance xylose metabolism in yeast.
Reference
Wei F., Li M., Wang M., et al. (2020). A C6/C5 co‐fermenting *Saccharomyces cerevisiae* strain with the alleviation of antagonism between xylose utilization and robustness. *GCB Bioenergy*, 13(1), 83–97.
Brat D., Boles E., Wiedemann B. (2009). Functional expression of a bacterial xylose isomerase in *Saccharomyces cerevisiae*. *Applied Microbiology and Biotechnology*, 75, 2304-2311.