Difference between revisions of "Part:BBa K2043005"
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<partinfo>BBa_K2043005 short</partinfo>
 
<partinfo>BBa_K2043005 short</partinfo>
  
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<b>Fabric Binding Domain 1 (BBa_K2043010) 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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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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The <b>Fabric Binding Domain</b> has affinity for Cotton, Silk, Polyester, Linen and Nylon. It is positively charged (+1) and proline rich. <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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<p>
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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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This part corresponds to Catechol-2,3-dioxygenase (<i>xylE</i>) fused to the Fabric Binding Domain 1 (BBa_K2043010) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction with EC number 1.13.11.2
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<img src="https://static.igem.org/mediawiki/parts/2/2f/Paris_Bettencourt_Catecholase_example.jpg">
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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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<b>Testing the part</b><br><br>
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The figure 1 shows of catechol 2,3-dioxygenases catalyzes the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds have structurally similarities to catechol, specially to the phenolic cycle of anthocyanins, making Catechol-2,3-dioxygenase a good candidate for anthocyanin degradation. <br>
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Catechol-2,3-dioxygenase is also found in many species of soil bacteria. This enzymes originally comes from <i>Pseudomonas putida </i>(NCBI Ref. Seq.: NP_542866.1), which we codon optimized for <i>E. Coli</i> and avoided the BsaI restriction sites. An His-tag was also added at the C-terminal. This tag allows for purification in an easier way.<br>
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We wanted to test Catechol-dioxygenases: one was 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 and we expected that the phenolic cycle of the anthocyanin could be a possible target for this enzyme. (Cerdan 1995, Kobayasi 1995 and Lin 2015). <br>
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The Fabric Binding Domain 1 (FBD1) has affinity for cotton, linen, polyester, silk and wool. It is positively charged (+1) and it is proline rich.<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 the activity of XylE-FBD1 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/5/50/Paris_Bettencourt_biobricks_xylE1.jpg <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/1/1f/Xyle_fbd1_shaded.jpg" width=600><br>
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Nonetheless, binding of the FBD1 in the N-terminal makes our protein unfunctional.
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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-FB1. The blue line represents the negative control, green line shows the activity of the cell extract containing XylE, and the red line corresponds with the cell extract  of cells expressing XylE-FBD.<br><br>
  
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<img src="https://static.igem.org/mediawiki/parts/5/50/Paris_Bettencourt_biobricks_xylE1.jpg" width=600><br>
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<span class='h3bb'>Sequence and Features</span>
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<b>Figure 3</b> XylE fusion proteins' activity was measured 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><br>
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<partinfo>BBa_K2043005 SequenceAndFeatures</partinfo>
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As the image indicates, there is a clear difference between our native and the control, but no between the fusion protein and the control. According with these results we can conclude that the enzyme lost the activity because the fusion peptide, or in the other hand that the addition of this peptide in N-terminal reduce the expression and therefore no XylE-FBD was present in the cell extract.<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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Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.
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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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<span class='h3bb'>Sequence and Features</span>
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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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<partinfo>BBa_K2043005 SequenceAndFeatures</partinfo>
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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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NCBI Reference Sequence: NP_542866.1
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Revision as of 16:02, 27 October 2016

xylE-FBD1 from Pseudomonas putida codon optimized for E. coli

This part corresponds to Catechol-2,3-dioxygenase (xylE) fused to the Fabric Binding Domain 1 (BBa_K2043010) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme catalyses the following chemical reaction with EC number 1.13.11.2


Figure 1 Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.

The figure 1 shows of catechol 2,3-dioxygenases catalyzes the extradiol ring-cleavage of catechol derivatives. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds have structurally similarities to catechol, specially to the phenolic cycle of anthocyanins, 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 enzymes originally comes from Pseudomonas putida (NCBI Ref. Seq.: NP_542866.1), which we codon optimized for E. Coli and avoided the BsaI restriction sites. An His-tag was also added at the C-terminal. This tag allows for purification in an easier way.
We wanted to test Catechol-dioxygenases: one was 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 and we expected that the phenolic cycle of the anthocyanin could be a possible target for this enzyme. (Cerdan 1995, Kobayasi 1995 and Lin 2015).
The Fabric Binding Domain 1 (FBD1) has affinity for cotton, linen, polyester, silk and wool. It is positively charged (+1) and it is proline rich.

Testing the part

We tested our cell extract for the activity of XylE-FBD1 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-FB1. The blue line represents the negative control, green line shows the activity of the cell extract containing XylE, and the red line corresponds with the cell extract of cells expressing XylE-FBD.


Figure 3 XylE fusion proteins' activity was measured 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.

As the image indicates, there is a clear difference between our native and the control, but no between the fusion protein and the control. According with these results we can conclude that the enzyme lost the activity because the fusion peptide, or in the other hand that the addition of this peptide in N-terminal reduce the expression and therefore no XylE-FBD was present in the cell extract.

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.

Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.

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 700
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]