Difference between revisions of "Part:BBa K5351001"
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<partinfo>BBa_K5351001 short</partinfo>
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<partinfo>BBa_K5351000 short</partinfo>
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<!-- 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>
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<partinfo>BBa_K5351001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K5351000 SequenceAndFeatures</partinfo>
  
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<!-- Uncomment this to enable Functional Parameter display  
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<!-- Uncomment this to enable Functional Parameter display
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K5351001 parameters</partinfo>
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<partinfo>BBa_K5351000 parameters</partinfo>
 
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= Origin =
 
= Origin =
  
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Synthesized by the team and constructed as a gRNA plasmid for yeast.
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E. coli; synthesized
  
 
<span id="properties"></span>
 
<span id="properties"></span>
 
= Properties =
 
= Properties =
  
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This part is designed to mutate the NFS1 gene in yeast, enhancing the metabolism of xylose, an essential step for efficient ethanol production.
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The plasmid pSCm-N20 is a genetic construct containing specific DNA sequences that enable the expression of a particular gene or genes in a host organism. It includes a promoter region for gene expression and possibly other regulatory elements necessary for the function of the inserted gene. It is used as the vector plasmid in our research.
  
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<span id="usage-and-biology"></span>
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<span id="cultivation-purification-sds-page"></span>
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= Usage and Biology =
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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.
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<span id="experimental-approach"></span>
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= Experimental Approach =
 
= Experimental Approach =
  
 
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/1.png" width="50%" style="display:block; margin:auto;" alt="Plasmid map of pSCm-N20" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 1. Plasmid map of pSCm-NFS1.</caption>
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         <caption>Figure 1. Plasmid map of pSCm-N20.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
</html>
 
</html>
  
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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.
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The plasmid pSCm was digested to obtain a linear backbone with a length of 5984 bp.
  
 
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<div style="text-align:center;">
 
<div style="text-align:center;">
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/2.jpg" width="50%" style="display:block; margin:auto;" alt="The gel electrophoresis validation of the linear pSCm-N20 plasmid backbone" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 2. Gel electrophoresis of pSCm-N20.</caption>
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         <caption>Figure 2. The gel electrophoresis validation of the linear pSCm-N20 plasmid backbone.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
</html>
 
</html>
  
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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.
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The band shown in Figure 2 indicates that the electrophoresis results are consistent with the expected plasmid size, confirming successful linearization of the plasmid backbone.
  
 
<html>
 
<html>
 
<div style="text-align:center;">
 
<div style="text-align:center;">
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/3.png" width="50%" style="display:block; margin:auto;" alt="The assembly of the pSCm-N20 plasmid with synthesized oligos" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 3. Transformation plate of pSCm-NFS1.</caption>
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         <caption>Figure 3. The assembly of the pSCm-N20 plasmid with synthesized oligos.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
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</html>
  
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Colonies were verified through colony PCR, and the correct fragment length of 288 bp was obtained.
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<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="Transformation plate of pSCm-N20" >
 +
    <div style="text-align:center;">
 +
        <caption>Figure 4. Transformation plate of pSCm-N20.</caption>
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    </div>
 +
</div>
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</html>
  
 
<html>
 
<html>
 
<div style="text-align:center;">
 
<div style="text-align:center;">
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/5.png" width="50%" style="display:block; margin:auto;" alt="Sequencing result of pSCm-N20" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 4. Gel electrophoresis validation of pSCm-NFS1.</caption>
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         <caption>Figure 5. Sequencing result of pSCm-N20.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
</html>
 
</html>
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Sequencing confirmed the successful construction of pSCm-NFS1, showing the correct sequence.
 
  
 
<html>
 
<html>
 
<div style="text-align:center;">
 
<div style="text-align:center;">
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/6.png" width="50%" style="display:block; margin:auto;" alt="PCR validation for integration of PsXI genes" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 5. Sequencing map of pSCm-NFS1.</caption>
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         <caption>Figure 6. PCR validation for integration of PsXI genes.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
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</html>
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The pSCm-NFS1 plasmid was introduced into yeast strains to induce mutations, leading to enhanced xylose metabolism.
 
  
 
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<div style="text-align:center;">
 
<div style="text-align:center;">
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/7.jpg" width="50%" style="display:block; margin:auto;" alt="PCR and colony map of strain xyl-8XI-NFS1" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 6. PCR verification of mutated strains.</caption>
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         <caption>Figure 7. PCR and colony map of strain xyl-8XI-NFS1.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
</html>
 
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Xylose metabolism capabilities were evaluated through a solid medium assay. Mutated strains demonstrated significantly improved xylose utilization compared to wild-type strains.
 
  
 
<html>
 
<html>
 
<div style="text-align:center;">
 
<div style="text-align:center;">
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/7.jpg" width="50%" style="display:block; margin:auto;" alt="Xylose metabolism plate assay" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/8.jpg" width="50%" style="display:block; margin:auto;" alt="PCR validation of strain xyl-8XI-ΔISU1" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 7. Xylose metabolism plate assay.</caption>
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         <caption>Figure 8. PCR validation of strain xyl-8XI-ΔISU1.</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
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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.
 
  
 
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/9.jpg" width="50%" style="display:block; margin:auto;" alt="PCR validation of strain xyl-8XI-nfs1-ΔISU1" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 8. HPLC results of ethanol production.</caption>
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         <caption>Figure 9. PCR validation of strain xyl-8XI-nfs1-ΔISU1.</caption>
 
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</div>
 
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/10.jpg" width="50%" style="display:block; margin:auto;" alt="Xylose metabolism analysis using plate assay" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 9. Colony PCR verification of xylose metabolism strains.</caption>
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         <caption>Figure 10. Xylose metabolism analysis using plate assay.</caption>
 
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The xylose utilization and ethanol production of the engineered strains were further validated through fermentation assays.
 
  
 
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     <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" >
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     <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 growth status of modified strains" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 10. Growth and ethanol production of xylose metabolism strains.</caption>
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         <caption>Figure 11. Comparison of xylose metabolism and growth status of modified strains.</caption>
 
     </div>
 
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The results confirm the success of genetic modifications to enhance xylose metabolism in yeast.
 
  
 
<html>
 
<html>
 
<div style="text-align:center;">
 
<div style="text-align:center;">
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     <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" >
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     <img src="https://static.igem.wiki/teams/5351/bba-k5351001/12.png" width="50%" style="display:block; margin:auto;" alt="Ethanol production levels of different strains" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
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         <caption>Figure 11. Comparison of xylose metabolism and ethanol production.</caption>
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         <caption>Table 1. Ethanol production levels of different strains (48 h).</caption>
 
     </div>
 
     </div>
 
</div>
 
</div>
 
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<span id="reference"></span>
 
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= Reference =
 
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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 09:47, 27 September 2024

pSCm-N20


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal NheI site found at 1135
    Illegal NheI site found at 4384
    Illegal NotI site found at 2401
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BamHI site found at 2246
    Illegal BamHI site found at 2357
    Illegal XhoI site found at 2408
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.


Origin

E. coli; synthesized

Properties

The plasmid pSCm-N20 is a genetic construct containing specific DNA sequences that enable the expression of a particular gene or genes in a host organism. It includes a promoter region for gene expression and possibly other regulatory elements necessary for the function of the inserted gene. It is used as the vector plasmid in our research.

Experimental Approach

Plasmid map of pSCm-N20
Figure 1. Plasmid map of pSCm-N20.

The plasmid pSCm was digested to obtain a linear backbone with a length of 5984 bp.

The gel electrophoresis validation of the linear pSCm-N20 plasmid backbone
Figure 2. The gel electrophoresis validation of the linear pSCm-N20 plasmid backbone.

The band shown in Figure 2 indicates that the electrophoresis results are consistent with the expected plasmid size, confirming successful linearization of the plasmid backbone.

The assembly of the pSCm-N20 plasmid with synthesized oligos
Figure 3. The assembly of the pSCm-N20 plasmid with synthesized oligos.

Transformation plate of pSCm-N20
Figure 4. Transformation plate of pSCm-N20.

Sequencing result of pSCm-N20
Figure 5. Sequencing result of pSCm-N20.

PCR validation for integration of PsXI genes
Figure 6. PCR validation for integration of PsXI genes.

PCR and colony map of strain xyl-8XI-NFS1
Figure 7. PCR and colony map of strain xyl-8XI-NFS1.

PCR validation of strain xyl-8XI-ΔISU1
Figure 8. PCR validation of strain xyl-8XI-ΔISU1.

PCR validation of strain xyl-8XI-nfs1-ΔISU1
Figure 9. PCR validation of strain xyl-8XI-nfs1-ΔISU1.

Xylose metabolism analysis using plate assay
Figure 10. Xylose metabolism analysis using plate assay.

Comparison of xylose metabolism and growth status of modified strains
Figure 11. Comparison of xylose metabolism and growth status of modified strains.

Ethanol production levels of different strains
Table 1. Ethanol production levels of different strains (48 h).