Part:BBa_K2043002
catA-FBD1 from Acinetobacter pittii, codon optimized for E. coli
This part corresponds to Catechol-1,2-dioxygenase 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 enzymes originally comes from Acinetobacter pittii, which we codon optimised for E. coli.
In order to facilitate working with this enzyme, we added a His-tag at the C-terminal. This tag allows for purification in an easier way.
The Fabric Binding Domain 1 (FBD1) has affinity for Cotton, Linnen, Polyester, Silk and Wool. It is positively charget (+1) and it is proline rich.
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.
In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.
Testing the part
We tested the activity of CatA-FBD1 using cell extract of cells expressing our protein.
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.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.
https://static.igem.org/mediawiki/parts/d/d5/Paris_Bettencourt_notebook_catA1_goodforsure.jpg
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.
Binding the FBD1 decreased slightly the activity of CatA, but the enzyme was still functional.
References
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.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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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