Difference between revisions of "Part:BBa K5477038"
 
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<partinfo>BBa_K5477038 short</partinfo>
 
<partinfo>BBa_K5477038 short</partinfo>
  
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In our system, the CYP3A4-pGAL1/10-POR composite part combines the CYP3A4 enzyme and cytochrome P450 oxidoreductase (POR) to form a highly efficient detoxification unit. The pGAL1/10 bidirectional promoter drives the expression of both CYP3A4 and POR simultaneously but in opposite directions. CYP3A4, a key phase I enzyme, is responsible for oxidizing around 50% of all clinically used drugs and environmental toxins. POR provides the electrons required for these oxidation reactions by transferring electrons from NADPH to CYP3A4, ensuring efficient metabolism of harmful xenobiotics and pharmaceuticals.
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The CYP3A4-pGAL1/10-POR composite part consists the CYP3A4 enzyme [https://parts.igem.org/Part:BBa_K5477020 BBa_K5477020] and cytochrome P450 oxidoreductase (POR) [https://parts.igem.org/Part:BBa_K5477022 BBa_K5477022] to form a detoxification module. The pGAL1/10 bidirectional promoter drives the co-expression of CYP3A4 and POR in opposite directions. CYP3A4, a phase I enzyme, is responsible for oxidizing around 50% of all clinically used drugs (1) (2) (3) (4). POR provides the electrons required for these oxidation reactions by transferring electrons from NADPH to CYP3A4 (5).
  
 
This 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.  
 
This 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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===Results===
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Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.
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Methodology: The detoxification module CYP1A1-pGAL1/10-POR with UDPD-pGAL1/10-UGT1A1 was induced with galactose and incubated with PCB. 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.
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All samples were filtered prior to being loaded into LC-MS glass vials.
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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 the 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>
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    <tr>
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      <th>Sample Number</th>
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      <th>Sample Name</th>
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      <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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    <tr>
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      <td>B1</td>
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      <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>B3</td>
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      <td>PCB standard in Absolute Ethanol</td>
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      <td>Standard</td>
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      <td>Black</td>
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    </tr>
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    <tr>
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      <td>B4</td>
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      <td>C1A1-U1A1 yeast + PCB pellet</td>
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      <td>Sample</td>
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      <td>Brown</td>
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    </tr>
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    <tr>
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      <td>B5</td>
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      <td>C1A1-U1A1 yeast + PCB media</td>
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      <td>Sample</td>
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      <td>Light pink</td>
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    </tr>
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    <tr>
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      <td>B6</td>
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      <td>Empty yeast + PCB pellet</td>
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      <td>Control for PCB incubation</td>
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      <td>Gray-Blue</td>
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    </tr>
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    <tr>
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      <td>B7</td>
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      <td>Empty yeast + PCB media</td>
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      <td>Control for PCB incubation</td>
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      <td>Dark Gray</td>
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    </tr>
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    <tr>
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      <td>B8</td>
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      <td>UGT2B15 yeast + BPA pellet</td>
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      <td>Sample</td>
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      <td>Lilac (Light Purple)</td>
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    </tr>
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    <tr>
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      <td>B9</td>
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      <td>UGT2B15 yeast + BPA media</td>
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      <td>Sample</td>
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      <td>Olive Green</td>
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    </tr>
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    <tr>
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      <td>B10</td>
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      <td>Empty yeast + BPA pellet</td>
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      <td>Control for BPA incubation</td>
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      <td>Light Gray</td>
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    </tr>
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    <tr>
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      <td>B11</td>
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      <td>UGT2B15 yeast strain</td>
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      <td>Control for detox system expression</td>
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      <td>Light Blue</td>
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    </tr>
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    <tr>
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      <td>B12</td>
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      <td>Empty yeast + BPA media</td>
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      <td>Control for BPA incubation</td>
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      <td>Yellow</td>
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    </tr>
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    <tr>
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      <td>B13</td>
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      <td>C3A4 + BPA + PCB pellet</td>
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      <td>Sample</td>
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      <td>Purple</td>
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    </tr>
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    <tr>
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      <td>B14</td>
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      <td>C3A4 + BPA + PCB media</td>
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      <td>Sample</td>
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      <td>Red</td>
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    </tr>
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    <tr>
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      <td>B15</td>
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      <td>C1A1-U1A1 yeast strain</td>
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      <td>Control for detox system expression</td>
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      <td>Light Green</td>
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    </tr>
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    <tr>
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      <td>B16</td>
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      <td>C3A4 yeast strain</td>
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      <td>Control for detox system expression</td>
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      <td>Royal Blue</td>
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    </tr>
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</table>
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Here, we tried to test CYP3A4 system on bith PCB and BPA. We decided to do so as CYP3A4 P450 enzyme is seen to work on a diverse substrates. However, here we were unable to find any unique peaks. Here the system was incubated with 10 µM of PCB#3 and BPA each.
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/results/compound-spec/pic1lcms-detox.png" width="700"></div></html>
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Figure 1 Base Peak Chromatogram for C3A4 detox system. Pellet, media, control samples are included.
  
  
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<partinfo>BBa_K5477038 parameters</partinfo>
 
<partinfo>BBa_K5477038 parameters</partinfo>
 
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===References===
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1. Bardal SK, Waechter JE, Martin DS. Chapter 6 - Pharmacogenetics. In: Bardal SK, Waechter JE, Martin DS, editors. Applied Pharmacology [Internet]. Philadelphia: W.B. Saunders; 2011. p. 53–8. Available from: https://www.sciencedirect.com/science/article/pii/B9781437703108000063
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2. Guengerich, F.. (2008). Cytochrome P450 and Chemical Toxicology. Chemical research in toxicology. 21. 70-83. 10.1021/tx700079z.
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3. Klyushova LS, Perepechaeva ML, Grishanova AY. The Role of CYP3A in Health and Disease. Biomedicines. 2022 Oct 24;10(11):2686. doi: 10.3390/biomedicines10112686. PMID: 36359206; PMCID: PMC9687714.
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4. Lynch T, Price A. The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am Fam Physician. 2007;76(3):391-396.
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 +
5. Pandey AV, Flück CE. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther. 2013;138(2):229-254. doi:10.1016/j.pharmthera.2013.01.010

Latest revision as of 13:18, 2 October 2024


CYP3A4-pGAL1/10-POR detox module against a wide array of contaminants


The CYP3A4-pGAL1/10-POR composite part consists the CYP3A4 enzyme BBa_K5477020 and cytochrome P450 oxidoreductase (POR) BBa_K5477022 to form a detoxification module. The pGAL1/10 bidirectional promoter drives the co-expression of CYP3A4 and POR in opposite directions. CYP3A4, a phase I enzyme, is responsible for oxidizing around 50% of all clinically used drugs (1) (2) (3) (4). POR provides the electrons required for these oxidation reactions by transferring electrons from NADPH to CYP3A4 (5).

This 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 CYP1A1-pGAL1/10-POR with UDPD-pGAL1/10-UGT1A1 was induced with galactose and incubated with PCB. 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 the 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.


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
B3 PCB standard in Absolute Ethanol Standard Black
B4 C1A1-U1A1 yeast + PCB pellet Sample Brown
B5 C1A1-U1A1 yeast + PCB media Sample Light pink
B6 Empty yeast + PCB pellet Control for PCB incubation Gray-Blue
B7 Empty yeast + PCB media Control for PCB incubation Dark Gray
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
B13 C3A4 + BPA + PCB pellet Sample Purple
B14 C3A4 + BPA + PCB media Sample Red
B15 C1A1-U1A1 yeast strain Control for detox system expression Light Green
B16 C3A4 yeast strain Control for detox system expression Royal Blue



Here, we tried to test CYP3A4 system on bith PCB and BPA. We decided to do so as CYP3A4 P450 enzyme is seen to work on a diverse substrates. However, here we were unable to find any unique peaks. Here the system was incubated with 10 µM of PCB#3 and BPA each.

Figure 1 Base Peak Chromatogram for C3A4 detox system. Pellet, media, control samples are included.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1483
    Illegal PstI site found at 2486
    Illegal PstI site found at 2683
    Illegal PstI site found at 3067
    Illegal PstI site found at 3507
    Illegal PstI site found at 3567
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2486
    Illegal PstI site found at 2683
    Illegal PstI site found at 3067
    Illegal PstI site found at 3507
    Illegal PstI site found at 3567
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1483
    Illegal PstI site found at 2486
    Illegal PstI site found at 2683
    Illegal PstI site found at 3067
    Illegal PstI site found at 3507
    Illegal PstI site found at 3567
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1483
    Illegal PstI site found at 2486
    Illegal PstI site found at 2683
    Illegal PstI site found at 3067
    Illegal PstI site found at 3507
    Illegal PstI site found at 3567
    Illegal NgoMIV site found at 2889
    Illegal NgoMIV site found at 3008
    Illegal AgeI site found at 1894
  • 1000
    COMPATIBLE WITH RFC[1000]



References

1. Bardal SK, Waechter JE, Martin DS. Chapter 6 - Pharmacogenetics. In: Bardal SK, Waechter JE, Martin DS, editors. Applied Pharmacology [Internet]. Philadelphia: W.B. Saunders; 2011. p. 53–8. Available from: https://www.sciencedirect.com/science/article/pii/B9781437703108000063

2. Guengerich, F.. (2008). Cytochrome P450 and Chemical Toxicology. Chemical research in toxicology. 21. 70-83. 10.1021/tx700079z.

3. Klyushova LS, Perepechaeva ML, Grishanova AY. The Role of CYP3A in Health and Disease. Biomedicines. 2022 Oct 24;10(11):2686. doi: 10.3390/biomedicines10112686. PMID: 36359206; PMCID: PMC9687714.

4. Lynch T, Price A. The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am Fam Physician. 2007;76(3):391-396.

5. Pandey AV, Flück CE. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther. 2013;138(2):229-254. doi:10.1016/j.pharmthera.2013.01.010