Difference between revisions of "Part:BBa K5477035"
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The composite part was cloned using the method of USER-cloning into YCp-H. YCp-H is a centromeric plasmid used in yeast that includes a HIS3 marker, allowing for selection in histidine auxotrophic yeast strains. Like other CEN plasmids, YCp-H contains a CEN sequence, ensuring that the plasmid replicates and segregates similarly to yeast chromosomes. This results in a low copy number (typically one to two copies per cell), providing stable maintenance of the plasmid.  
 
The composite part was cloned using the method of USER-cloning into YCp-H. YCp-H is a centromeric plasmid used in yeast that includes a HIS3 marker, allowing for selection in histidine auxotrophic yeast strains. Like other CEN plasmids, YCp-H contains a CEN sequence, ensuring that the plasmid replicates and segregates similarly to yeast chromosomes. This results in a low copy number (typically one to two copies per cell), providing stable maintenance of the plasmid.  
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 +
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===Results===
 +
 +
Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.
 +
 +
Methodology: The detoxification module - UGT2B15 - GAL1,10 – UDPD was induced with galactose and incubated with BPA. Following overnight incubation, each detoxification system was centrifuged in Eppendorf tubes to separate the cells from the supernatant. The supernatant, henceforth referred to as the “media” samples, was collected. Absolute ethanol was added to the cell pellets, which were vortexed with micro glass beads to lyse the cells. The mixtures were centrifuged to separate cellular debris from the lysate, and the resulting supernatant was collected as the “pellet” samples.
 +
 +
All samples were filtered prior to being loaded into LC-MS glass vials.
 +
 +
Result: We did not have optimized LC-MS methods available for detecting highly hydrophobic compounds, even with the use of a Reverse Phase Column. Additionally, it took some time to identify ethanol as a suitable solvent for the system available in our department. Therefore, the results presented here are based on data generated from a single run of the samples on the LC-MS system. A previous run, where DMSO was used as a solvent, was excluded from the experiment due to the presence of multiple nonspecific peaks.
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<table>
 +
    <tr>
 +
      <th>Sample Number</th>
 +
      <th>Sample Name</th>
 +
      <th>Purpose</th>
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      <th>Colour in Chromatogram (also mentioned in chromatogram legend)</th>
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    </tr>
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  </thead>
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  <tbody>
 +
    <tr>
 +
      <td>B1</td>
 +
      <td>BPA standard in Absolute Ethanol</td>
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      <td>Standard</td>
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      <td>Ochre</td>
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    </tr>
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    <tr>
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      <td>B2</td>
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      <td>Empty yeast strain</td>
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      <td>Control for yeast strain</td>
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      <td>Dark Green</td>
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    </tr>
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    <tr>
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      <td>B8</td>
 +
      <td>UGT2B15 yeast + BPA pellet</td>
 +
      <td>Sample</td>
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      <td>Lilac (Light Purple)</td>
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    </tr>
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    <tr>
 +
      <td>B9</td>
 +
      <td>UGT2B15 yeast + BPA media</td>
 +
      <td>Sample</td>
 +
      <td>Olive Green</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B10</td>
 +
      <td>Empty yeast + BPA pellet</td>
 +
      <td>Control for BPA incubation</td>
 +
      <td>Light Gray</td>
 +
    </tr>
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    <tr>
 +
      <td>B11</td>
 +
      <td>UGT2B15 yeast strain</td>
 +
      <td>Control for detox system expression</td>
 +
      <td>Light Blue</td>
 +
    </tr>
 +
    <tr>
 +
      <td>B12</td>
 +
      <td>Empty yeast + BPA media</td>
 +
      <td>Control for BPA incubation</td>
 +
      <td>Yellow</td>
 +
    </tr>
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</table>
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/ugt2b15-pellet-media-control-1.png" width="400"></div></html>
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Figure X Base Peak Chromatogram for UGT2B15 detox system. Pellet, media, control samples are included
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The Base Peak Chromatogram for UGT2B15 detox system shows the peak for UGT2B15 yeast + BPA pellet (B8, light purple) between retention time 20 and 30 minutes. The same peaks are seen for empty yeast + BPA pellet (B10, light gray). These intesne peak look like hydrophobic parts of the cell debris.
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 +
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/ugt2b15-zoomed.png" width="400"></div></html>
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Figure X Base Peak Chromatogram for UGT2B15 detox system zoomed in to 17.5 minutes to 21.0 minutes retention time.
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/ugt2b15-zoomed-2.png" width="400"></div></html>
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Figure X Base Peak Chromatogram for UGT2B15 detox system zoomed in to 9.6 minutes to 10.8 minutes retention time.
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 +
The peak at retention time 10 minutes was one of the common peaks seen in all samples apart from BPA standard. This could be a signal given by yeast cells in general.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 03:25, 2 October 2024


UDPD-pGAL1/10-UGT2B15 detox module against BPA


This composite part consists of UDP-glucuronosyltransferase 2B15 (UGT2B15) BBa_K54770169 , pGAL1/10 bidirectional promoter BBa_K5477005 and UDP-glucose dehydrogenase BBa_K5477018. pGAL1/10 drives the expression of the two enzymes in the opposite direction. UDPD synthesizes the necessary precursor (UDP-glucoronic acid) that UGT2B15 uses for phase II detoxification of BPA (1) (2).

The composite part was cloned using the method of USER-cloning into YCp-H. YCp-H is a centromeric plasmid used in yeast that includes a HIS3 marker, allowing for selection in histidine auxotrophic yeast strains. Like other CEN plasmids, YCp-H contains a CEN sequence, ensuring that the plasmid replicates and segregates similarly to yeast chromosomes. This results in a low copy number (typically one to two copies per cell), providing stable maintenance of the plasmid.


Results

Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.

Methodology: The detoxification module - UGT2B15 - GAL1,10 – UDPD was induced with galactose and incubated with BPA. Following overnight incubation, each detoxification system was centrifuged in Eppendorf tubes to separate the cells from the supernatant. The supernatant, henceforth referred to as the “media” samples, was collected. Absolute ethanol was added to the cell pellets, which were vortexed with micro glass beads to lyse the cells. The mixtures were centrifuged to separate cellular debris from the lysate, and the resulting supernatant was collected as the “pellet” samples.

All samples were filtered prior to being loaded into LC-MS glass vials.

Result: We did not have optimized LC-MS methods available for detecting highly hydrophobic compounds, even with the use of a Reverse Phase Column. Additionally, it took some time to identify ethanol as a suitable solvent for the system available in our department. Therefore, the results presented here are based on data generated from a single run of the samples on the LC-MS system. A previous run, where DMSO was used as a solvent, was excluded from the experiment due to the presence of multiple nonspecific peaks.

</thead> <tbody>
Sample Number Sample Name Purpose Colour in Chromatogram (also mentioned in chromatogram legend)
B1 BPA standard in Absolute Ethanol Standard Ochre
B2 Empty yeast strain Control for yeast strain Dark Green
B8 UGT2B15 yeast + BPA pellet Sample Lilac (Light Purple)
B9 UGT2B15 yeast + BPA media Sample Olive Green
B10 Empty yeast + BPA pellet Control for BPA incubation Light Gray
B11 UGT2B15 yeast strain Control for detox system expression Light Blue
B12 Empty yeast + BPA media Control for BPA incubation Yellow


Figure X Base Peak Chromatogram for UGT2B15 detox system. Pellet, media, control samples are included

The Base Peak Chromatogram for UGT2B15 detox system shows the peak for UGT2B15 yeast + BPA pellet (B8, light purple) between retention time 20 and 30 minutes. The same peaks are seen for empty yeast + BPA pellet (B10, light gray). These intesne peak look like hydrophobic parts of the cell debris.


Figure X Base Peak Chromatogram for UGT2B15 detox system zoomed in to 17.5 minutes to 21.0 minutes retention time.


Figure X Base Peak Chromatogram for UGT2B15 detox system zoomed in to 9.6 minutes to 10.8 minutes retention time.

The peak at retention time 10 minutes was one of the common peaks seen in all samples apart from BPA standard. This could be a signal given by yeast cells in general.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3466
    Illegal PstI site found at 542
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3466
    Illegal PstI site found at 542
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3466
    Illegal BglII site found at 1122
    Illegal BamHI site found at 3286
    Illegal BamHI site found at 3580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3466
    Illegal PstI site found at 542
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3466
    Illegal PstI site found at 542
    Illegal AgeI site found at 1828
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Hanioka N, Naito T, Narimatsu S. Human UDP-glucuronosyltransferase isoforms involved in bisphenol A glucuronidation. Chemosphere. 2008 Dec 1;74(1):33–6.

2. Oka T, Jigami Y. Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae. FEBS J. 2006 Jun;273(12):2645–57.