Difference between revisions of "Part:BBa K2043003"
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<partinfo>BBa_K2043003 short</partinfo>
 
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This part corresponds to <b>Catechol-1,2-dioxygenase</b> fused to the Fabric Binding Domain 1 (BBa_K2043017) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from <i>Acinetobacter pittii</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 10</b> (FBD10) has affinity for Cellulose. It is positively charged (+1).<br><br>
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This part corresponds to Catechol-1,2-dioxygenase (<i>catA</i> <a href="https://parts.igem.org/Part:BBa_K2043001">BBa_K2043001</a>) fused to the Fabric Binding Domain 10 (FBD10, <a href="https://parts.igem.org/Part:BBa_K2043017">BBa_K2043017</a>) 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 is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol, EC number 1.13.11.1. The fabric domain was fused to the N-terminal end.
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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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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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We took the CatA enzyme from <i>Acinetobacter pittii</i> (NCBI Ref. Seq.: YP_004995593.1), which we codon optimized for <i>E. coli</i> while removing the BsaI restriction sites. A His-tag was added to the C-terminal end for protein purification.<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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<b>Testing the part</b><br><br>
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We wanted to test Catechol-dioxygenases as potential anthocyanin degrading enzymes: to this end, we tested CatA from <i>Acinetobacter pittii</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). <br>
  
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We tested the activity of CatA-FBD10 using cell extract of cells expressing our protein. <br>
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<b>The Fabric Binding Domain 10 (FBD10)</b> has affinity for cellulose and has a positve charge (+1). We fused FBD10 to the N-terminus of CatA with the aim of improving its ability to bind to and degrade fabric stains.
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We tested our cell extract for CatA activity in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. <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/b/b1/Paris_Bettencourt_biobricks_catA10.jpg <br><br>
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As the image indicates, there is a clear difference between our native and fusion enzymes and the control. We measured the reaction product at 260nm, which results from the oxidation of Catechol. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional.<br>
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<h2>Testing the part</h2>
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Binding the FBD1 decreased slightly the activity of CatA, but the enzyme was still functional.
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We tested the activity and expression of CatA-FBD10 using cell extract from transformants of <i>E. coli</i> overexpressing the protein to determine the effect of adding the FBD. We wanted to observe whether the fusion of FBD10 would affect the activity, or the expression of the protein. Since the FBD10 was fused at the N-terminal end of the protein, close to the promoter, it might affect the folding of the protein and expression.<br>
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<img src="https://static.igem.org/mediawiki/parts/b/b1/Paris_Bettencourt_biobricks_catA10.jpg" width=600><br>
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<span class='h3bb'>Sequence and Features</span>
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<b>Figure 2</b> CatA fusion protein activity was measured in Sodium Phosphate 50mM at pH 7, with 30 mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, when the substrate had been completely consumed by the native protein. Control corresponds to non CatA-expressing cells.
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<partinfo>BBa_K2043003 SequenceAndFeatures</partinfo>
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We observed a clear difference between our native and fusion enzymes and the control (Figure 2). We measured the reaction product, cis,cis-muconic acid, at 260nm. Since the reaction product is produced at higher levels from cells expressing CatA than in the control, we can confirm that the enzyme was functional, even though activity is slightly decreased compared to the protein without FBD10.
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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.<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.<br><br>
  
 
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>
 
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>
  
 
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>
 
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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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.<br><br>
  
 
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>
 
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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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: YP_004995593.1
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2043002 SequenceAndFeatures</partinfo>

Latest revision as of 15:38, 27 October 2016

catA-FBD10 from Acinetobacter pittii, codon optimized for E.coli

This part corresponds to Catechol-1,2-dioxygenase (catA BBa_K2043001) fused to the Fabric Binding Domain 10 (FBD10, BBa_K2043017) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol, EC number 1.13.11.1. The fabric domain was fused to the N-terminal end.


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

We took the CatA enzyme from Acinetobacter pittii (NCBI Ref. Seq.: YP_004995593.1), which we codon optimized for E. coli while removing the BsaI restriction sites. A His-tag was added to the C-terminal end for protein purification.
We wanted to test Catechol-dioxygenases as potential anthocyanin degrading enzymes: to this end, we tested CatA from Acinetobacter pittii, 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).
The Fabric Binding Domain 10 (FBD10) has affinity for cellulose and has a positve charge (+1). We fused FBD10 to the N-terminus of CatA with the aim of improving its ability to bind to and degrade fabric stains.

Testing the part

We tested the activity and expression of CatA-FBD10 using cell extract from transformants of E. coli overexpressing the protein to determine the effect of adding the FBD. We wanted to observe whether the fusion of FBD10 would affect the activity, or the expression of the protein. Since the FBD10 was fused at the N-terminal end of the protein, close to the promoter, it might affect the folding of the protein and expression.


Figure 2 CatA fusion protein activity was measured in Sodium Phosphate 50mM at pH 7, with 30 mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, when the substrate had been completely consumed by the native protein. Control corresponds to non CatA-expressing cells.

We observed a clear difference between our native and fusion enzymes and the control (Figure 2). We measured the reaction product, cis,cis-muconic acid, at 260nm. Since the reaction product is produced at higher levels from cells expressing CatA than in the control, we can confirm that the enzyme was functional, even though activity is slightly decreased compared to the protein without FBD10.

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.

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.

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.

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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]