Difference between revisions of "Part:BBa K5351001"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K5351001 short</partinfo>
 
<partinfo>BBa_K5351001 short</partinfo>
  
−
pSCm-NFS1mu
 
  
  
−
<!-- Add more about the biology of this part here
+
<!-- Add more about the biology of this part here -->
−
===Usage and Biology===
+
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5351001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5351001 SequenceAndFeatures</partinfo>
−
 
  
 
<!-- 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>
−
<!-- -->
+
-->
 +
 
 +
__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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1135
    Illegal NheI site found at 4384
    Illegal NotI site found at 2401
  • 21
    INCOMPATIBLE 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
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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

Plasmid map of pSCm-NFS1
Figure 1. Plasmid map of pSCm-NFS1.

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.

Gel electrophoresis of pSCm-N20
Figure 2. Gel electrophoresis of pSCm-N20.

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.

Transformation plate of pSCm-NFS1
Figure 3. Transformation plate of pSCm-NFS1.

Colonies were verified through colony PCR, and the correct fragment length of 288 bp was obtained.

Gel electrophoresis validation of pSCm-NFS1
Figure 4. Gel electrophoresis validation of pSCm-NFS1.

Sequencing confirmed the successful construction of pSCm-NFS1, showing the correct sequence.

Sequencing map of pSCm-NFS1
Figure 5. Sequencing map of pSCm-NFS1.

The pSCm-NFS1 plasmid was introduced into yeast strains to induce mutations, leading to enhanced xylose metabolism.

PCR verification of mutated strains
Figure 6. PCR verification of mutated strains.

Xylose metabolism capabilities were evaluated through a solid medium assay. Mutated strains demonstrated significantly improved xylose utilization compared to wild-type strains.

Xylose metabolism plate assay
Figure 7. Xylose metabolism plate assay.

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.

HPLC results of ethanol production
Figure 8. HPLC results of ethanol production.

Colony PCR verification of xylose metabolism strains
Figure 9. Colony PCR verification of xylose metabolism strains.

The xylose utilization and ethanol production of the engineered strains were further validated through fermentation assays.

Growth and ethanol production of xylose metabolism strains
Figure 10. Growth and ethanol production of xylose metabolism strains.

The results confirm the success of genetic modifications to enhance xylose metabolism in yeast.

Comparison of xylose metabolism and ethanol production
Figure 11. Comparison of xylose metabolism and ethanol production.

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.