Difference between revisions of "Part:BBa K2043006"
 
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<partinfo>BBa_K2043006 short</partinfo>
 
<partinfo>BBa_K2043006 short</partinfo>
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<b>Fabric Binding Domain 10 (BBa_K2043017) in the N-terminal</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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<p>This part corresponds to Catechol-2,3-dioxygenase (<i>xylE</i>), EC number 1.13.11.2, fused to Fabric Binding Domain 10 (BBa_K2043017) 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 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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<br>
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The <b>Fabric Binding Domain</b> has affinity for cellulose. It is positively charged (+1) . <br><br>
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<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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<br>
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<b>Figure 1</b> Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.<br><br>
  
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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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Figure 1 shows catechol 2,3-dioxygenases catalyzing the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds have structural similarities to catechol, specially the possession of a phenolic ring. <br>
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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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Catechol-2,3-dioxygenase is also found in many species of soil bacteria. We took this enzyme from <i>Pseudomonas putida </i>(NCBI Ref. Seq.: NP_542866.1), codon optimized the gene for <i>E. coli</i> while removing the BsaI restriction sites. A His-tag was also added at the C-terminal for protein purification.<br>
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XylE was one of two Catechol-dioxygenases we tested, in addition to CatA (BBa_K2043001). 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). <br>
  
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<b>Testing the part</b><br><br>
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The Fabric Binding Domain 10 (FBD10) has affinity for Cellulose. It is positively charged (+1) <br><br>
  
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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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We tested our cell extract for XylE activity in Potassium Phosphate 100mM at pH 7.5, with 30mM of 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 our cell extract for XylE as for XylE-FBD10 activity in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Control corresponds to cells which do not express our proteins. In all cases, values measured correspond to reaction product, 2-hydroxymuconate semialdehyde.<br><br>
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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/f/f5/Paris_Bettencourt-biobricks_xylE10.png <br><br>
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As the image indicates, there is a clear difference between our native 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>
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<img src="https://static.igem.org/mediawiki/parts/5/5e/Xyle_fbd10_shaded.jpg" width=600><br>
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he binding of the FBD10 had a negative effect on the enzymatic activity, but nonetheless the activity of the enzyme was still observed.
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<b>Figure 2</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 and XylE-FB10. The blue line represents the negative control, the green line shows the activity of the cell extract containing XylE, and the red line corresponds with the cell extract  of cells expressing XylE-FBD10.<br><br>
  
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<img src="https://static.igem.org/mediawiki/parts/f/f5/Paris_Bettencourt-biobricks_xylE10.png" width=600><br>
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<span class='h3bb'>Sequence and Features</span>
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<b>Figure 3</b> XylE fusion protein activity. Measurements were taken after 12 min, at which time the substrate had been completely consumed.<br><br>
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<partinfo>BBa_K2043011 SequenceAndFeatures</partinfo>
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As the image indicates, there is a clear difference between the native protein and the control, and also between the fusion protein and the control. Based on these results, we can conclude that the enzyme has activity, however it has been reduced sustanctialy respect to the wild type.<br><br>
  
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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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<h2>References</h2>
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Boyer, S., Biswas, D., Soshee, A. K., Scaramozzino, N., Nizak, C., & Rivoire, O. (2016). Hierarchy and extremes in selections from pools of randomized proteins. Proceedings of the National Academy of Sciences, 201517813.
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<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.
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<br><br>
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Jain, P., Soshee, A., Narayanan, S. S., Sharma, J., Girard, C., Dujardin, E., & Nizak, C. (2014). Selection of arginine-rich anti-gold antibodies engineered for plasmonic colloid self-assembly. The Journal of Physical Chemistry C, 118(26), 14502-14510.
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<br><br>
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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.
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<br><br>
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Soshee, A., Zürcher, S., Spencer, N. D., Halperin, A., & Nizak, C. (2013). General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules, 15(1), 113-121.
  
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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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</p>
  
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Francisco, J. A., Stathopoulos, C., Warren, R. A., Kilburn, D. G., & Georgiou, G. (1993). Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains. Bio/technology (Nature Publishing Company), 11(4), 491-495.<br><br>
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</html>
  
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Jain, P., Soshee, A., Narayanan, S. S., Sharma, J., Girard, C., Dujardin, E., & Nizak, C. (2014). Selection of arginine-rich anti-gold antibodies engineered for plasmonic colloid self-assembly. The Journal of Physical Chemistry C, 118(26), 14502-14510.<br><br>
 
  
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Soshee, A., Zürcher, S., Spencer, N. D., Halperin, A., & Nizak, C. (2013). General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules, 15(1), 113-121.<br><br>
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<span class='h3bb'>Sequence and Features</span>
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Boyer, S., Biswas, D., Soshee, A. K., Scaramozzino, N., Nizak, C., & Rivoire, O. (2016). Hierarchy and extremes in selections from pools of randomized proteins. Proceedings of the National Academy of Sciences, 201517813.<br><br>
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<partinfo>BBa_K2043006 SequenceAndFeatures</partinfo>
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NCBI Reference Sequence: NP_542866.1
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Latest revision as of 17:13, 27 October 2016


xylE-FBD10 from Pseudomonas putida codon optimized for E. coli

This part corresponds to Catechol-2,3-dioxygenase (xylE), EC number 1.13.11.2, fused to Fabric Binding Domain 10 (BBa_K2043017) 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 Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.

Figure 1 shows catechol 2,3-dioxygenases catalyzing the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds have structural similarities to catechol, specially the possession of a phenolic ring.
Catechol-2,3-dioxygenase is also found in many species of soil bacteria. We took this enzyme from Pseudomonas putida (NCBI Ref. Seq.: NP_542866.1), codon optimized the gene for E. coli while removing the BsaI restriction sites. A His-tag was also added at the C-terminal for protein purification.
XylE was one of two Catechol-dioxygenases we tested, in addition to CatA (BBa_K2043001). 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).
The Fabric Binding Domain 10 (FBD10) has affinity for Cellulose. It is positively charged (+1)

Testing the part

We tested our cell extract for XylE as for XylE-FBD10 activity in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Control corresponds to cells which do not express our proteins. In all cases, values measured correspond to reaction product, 2-hydroxymuconate semialdehyde.


Figure 2 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 and XylE-FB10. The blue line represents the negative control, the green line shows the activity of the cell extract containing XylE, and the red line corresponds with the cell extract of cells expressing XylE-FBD10.


Figure 3 XylE fusion protein activity. Measurements were taken after 12 min, at which time the substrate had been completely consumed.

As the image indicates, there is a clear difference between the native protein and the control, and also between the fusion protein and the control. Based on these results, we can conclude that the enzyme has activity, however it has been reduced sustanctialy respect to the wild type.

References

Boyer, S., Biswas, D., Soshee, A. K., Scaramozzino, N., Nizak, C., & Rivoire, O. (2016). Hierarchy and extremes in selections from pools of randomized proteins. Proceedings of the National Academy of Sciences, 201517813.

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.

Jain, P., Soshee, A., Narayanan, S. S., Sharma, J., Girard, C., Dujardin, E., & Nizak, C. (2014). Selection of arginine-rich anti-gold antibodies engineered for plasmonic colloid self-assembly. The Journal of Physical Chemistry C, 118(26), 14502-14510.

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.

Soshee, A., Zürcher, S., Spencer, N. D., Halperin, A., & Nizak, C. (2013). General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules, 15(1), 113-121.


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 703
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]