Difference between revisions of "Part:BBa K2043004"
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<partinfo>BBa_K2043004 short</partinfo>
 
<partinfo>BBa_K2043004 short</partinfo>
  
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This part corresponds to <b>Catechol-2,3-dioxygenase</b> cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from <i>Pseudomonas putida</i>, which we <b>codon optimised for <i>E. coli</i></b>.<br>
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In order to facilitate working with this enzyme, we added a <b>His-tag</b> at the <b>C-terminal</b>. This tag allows for purification in an easier way.<br><br>
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<h2>Description</h2>
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We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us. <br>
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This part corresponds to the gene coding for the protein Catechol-2,3-dioxygenase (<i>xylE</i>), EC number 1.13.11.2, cloned by the Paris Bettencourt team in 2016 in the context of the <a href="http://2016.igem.org/Team:Paris_Bettencourt">Frank&Stain project</a>. This enzyme catalyses the following chemical reaction:
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In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.<br><br>
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<img src="https://static.igem.org/mediawiki/parts/a/a0/2016_paris_bettencourt_xylE_mechanism.png" width=400>
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<b>Figure 1</b> oxidation of catechol to 2-hydroxymuconate semialdehyde taken from wikipedia commons, created by user Yikrazuul.<br><br>
  
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<b>Testing the part</b><br><br>
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Figure 1 shows catechol 2,3-dioxygenase catalysis of the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-2,3-dioxygenase a good candidate for anthocyanin degradation. Catechol-2,3-dioxygenase is also found in many species of soil bacteria. This enzyme originally comes from <i>Pseudomonas putida</i> (NCBI Ref. Seq.: NP_542866.1), which we codon optimized for <i>E. Coli</i> while removing BsaI restriction sites. A His-tag was also added at the C-terminal end for protein purification. <br>
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We wanted to test Catechol-dioxygenases: in addition to CatA (BBa_K2043001), we tested XylE from <i>Pseudomonas putida</i> , which uses catechol as a main substrate. We hypothesized that this enzyme would be a strong candidate for removal of red wine stains because catechol shares important structural similarities with anthocyanin (Cerdan 1995, Kobayasi 1995 and Lin 2015).
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</p>
  
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We tested the activity of XylE using cell extract of cells expressing our protein. <br>
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<h2>Testing the part</h2>
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First, we performed an SDS-PAGE to check whether the protein was being expressed. <br><br>
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https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg
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<br><br>
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The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity. <br>
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<p>
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We tested our cell extract for XylE activity in Potassium Phosphate 100mM at pH 7.5, with 30mM os Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed. <br>
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We tested the expression and activity of XylE using cell extract from <i>E. coli</i> overexpressing the protein. First, we performed an SDS-PAGE to check whether the protein was successfully expressed.
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Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product. <br><br>
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https://static.igem.org/mediawiki/parts/8/82/Paris_Bettencourt_notebook_xylE_good.jpg
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As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 475nm, which results from the oxidation of Catechol. Since much more reaction product is produced with cells expressing XylE than in the control, we can affirm that the enzyme was functional. <br><br>
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<img src="https://static.igem.org/mediawiki/parts/1/1e/Paris_Bettencourt_notebook_GELS.jpg" width=800><br>
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<b>Figure 2</b> SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: <i>E.coli </i> strain BL21(DE3), expressing XylE, was induced for 5 hours with 0.5mM IPTG. After 5 hours, the OD (600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmli sample buffer (from BIO-RAD) to a final OD600 of 10. The cells were cooked at 95ºC for 10 min, and 10 uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoscope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking. As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa. <br><br>
  
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We then tested for XylE protein activity using the cell extract in Sodium Phosphate 50mM at pH 7.5, with 30 mM of Catechol as substrate, as recommended in the literature (Cerdan, 1995; Kobayashi, 1995). Control corresponds to cells not expressing XylE. In all cases, values measured correspond to reaction product.<br><br>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2043011 SequenceAndFeatures</partinfo>
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<img src="https://static.igem.org/mediawiki/parts/1/1a/Xyle_line.jpg" width=600><br>
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<b>Figure 3</b> Absorbance of the reaction product, 2-hydroxymunonic semialdehyde. The absorbance of the product was measured at 375nm over a period of time in order to follow the activity of the reaction of XylE. The blue line represents the negative control, and the green line shows the activity of the cell extract containing XylE. <br><br>
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<img src="https://static.igem.org/mediawiki/parts/8/82/Paris_Bettencourt_notebook_xylE_good.jpg" width=600><br>
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<b>Figure 4</b> This barplot is the result of the analysis of the data presented in Figure 3. XylE activity was measured with 30 mM of Catechol as substrate. <br> <br>
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As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product, (2-hydroxymunonic semialdehyde), at 375nm. Since much more reaction product is produced with cells expressing XylE than in the control, we can affirm that the enzyme was functional. <br> <br>
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<h2>References</h2>
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Cerdan, P., Rekik, M., & Harayama, S. (1995). Substrate Specificity Differences Between Two Catechol 2, 3‐Dioxygenases Encoded by the TOL and NAH Plasmids from Pseudomonas putida. European journal of biochemistry, 229(1), 113-118.<br><br>
  
 
Kobayashi, T., Ishida, T., Horiike, K., Takahara, Y., Numao, N., Nakazawa, A., ... & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2, 3-dioxygenase with high specific activity by genetically engineered Escherichia coli. Journal of biochemistry, 117(3), 614-622. <br><br>
 
Kobayashi, T., Ishida, T., Horiike, K., Takahara, Y., Numao, N., Nakazawa, A., ... & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2, 3-dioxygenase with high specific activity by genetically engineered Escherichia coli. Journal of biochemistry, 117(3), 614-622. <br><br>
  
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Cerdan, P., Rekik, M., & Harayama, S. (1995). Substrate Specificity Differences Between Two Catechol 2, 3‐Dioxygenases Encoded by the TOL and NAH Plasmids from Pseudomonas putida. European journal of biochemistry, 229(1), 113-118. <br><br>
 
  
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NCBI Reference Sequence: NP_542866.1
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2043004 SequenceAndFeatures</partinfo>

Latest revision as of 16:07, 27 October 2016

xylE from Pseudomonas putida codon optimized for E. coli

Description

This part corresponds to the gene coding for the protein Catechol-2,3-dioxygenase (xylE), EC number 1.13.11.2, cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction:


Figure 1 oxidation of catechol to 2-hydroxymuconate semialdehyde taken from wikipedia commons, created by user Yikrazuul.

Figure 1 shows catechol 2,3-dioxygenase catalysis of the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-2,3-dioxygenase a good candidate for anthocyanin degradation. Catechol-2,3-dioxygenase is also found in many species of soil bacteria. This enzyme originally comes from Pseudomonas putida (NCBI Ref. Seq.: NP_542866.1), which we codon optimized for E. Coli while removing BsaI restriction sites. A His-tag was also added at the C-terminal end for protein purification.
We wanted to test Catechol-dioxygenases: in addition to CatA (BBa_K2043001), we tested XylE from Pseudomonas putida , which uses catechol as a main substrate. We hypothesized that this enzyme would be a strong candidate for removal of red wine stains because catechol shares important structural similarities with anthocyanin (Cerdan 1995, Kobayasi 1995 and Lin 2015).

Testing the part

We tested the expression and activity of XylE using cell extract from E. coli overexpressing the protein. First, we performed an SDS-PAGE to check whether the protein was successfully expressed.
Figure 2 SDS-PAGE gels for expression of BpuI, CatA and XylE. Sample preparation: E.coli strain BL21(DE3), expressing XylE, was induced for 5 hours with 0.5mM IPTG. After 5 hours, the OD (600nm) was measured, the cells centrifuged and the pellet was resuspended in Laemmli sample buffer (from BIO-RAD) to a final OD600 of 10. The cells were cooked at 95ºC for 10 min, and 10 uL of the resulting solution were loaded on the gel. Ladder used: Kaleidoscope. Staining: The gel was washed 3x with miliQ water to remove the SDS and staining was performed using BioRad Comassie for 30min. De-staining was performed by leaving the gel in miliQ water for 1 hour with gentle shaking. As expected, no overexpression was observed in BL21(DE3), whether it was induced or not induced. BpuI was correctly overexpressed, with the observed size being the expected, around 59kDa. CatA overexpression was very mild, but the correct sized band was observed, of around 34kDa. XylE was correctly overexpressed, with the correct band being observed, of around 36kDa.

We then tested for XylE protein activity using the cell extract in Sodium Phosphate 50mM at pH 7.5, with 30 mM of Catechol as substrate, as recommended in the literature (Cerdan, 1995; Kobayashi, 1995). Control corresponds to cells not expressing XylE. In all cases, values measured correspond to reaction product.


Figure 3 Absorbance of the reaction product, 2-hydroxymunonic semialdehyde. The absorbance of the product was measured at 375nm over a period of time in order to follow the activity of the reaction of XylE. The blue line represents the negative control, and the green line shows the activity of the cell extract containing XylE.


Figure 4 This barplot is the result of the analysis of the data presented in Figure 3. XylE activity was measured with 30 mM of Catechol as substrate.

As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product, (2-hydroxymunonic semialdehyde), at 375nm. Since much more reaction product is produced with cells expressing XylE than in the control, we can affirm that the enzyme was functional.

References

Cerdan, P., Rekik, M., & Harayama, S. (1995). Substrate Specificity Differences Between Two Catechol 2, 3‐Dioxygenases Encoded by the TOL and NAH Plasmids from Pseudomonas putida. European journal of biochemistry, 229(1), 113-118.

Kobayashi, T., Ishida, T., Horiike, K., Takahara, Y., Numao, N., Nakazawa, A., ... & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2, 3-dioxygenase with high specific activity by genetically engineered Escherichia coli. Journal of biochemistry, 117(3), 614-622.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 670
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]