Difference between revisions of "Part:BBa K2043002"

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	<img src="https://static.igem.org/mediawiki/parts/d/d5/Paris_Bettencourt_notebook_catA1_goodforsure.jpg" width=600><br>		<img src="https://static.igem.org/mediawiki/parts/d/d5/Paris_Bettencourt_notebook_catA1_goodforsure.jpg" width=600><br>
−	<b>Figure 2</b>CatA fusion protein activity was measured in Sodium Phosphate 50 mM at pH 7, with 30 mM of Catechol substrate according to previously established techniques (Lin 2015). Measurements were taken after 35 min, at which time the substrate had been completely consumed by the native protein. Control corresponds to cells to non-CatA expressing cells.	+	<b>Figure 2</b> CatA fusion protein activity was measured in Sodium Phosphate 50 mM at pH 7, with 30 mM of Catechol substrate according to previously established techniques (Lin 2015). Measurements were taken after 35 min, at which time the substrate had been completely consumed by the native protein. Control corresponds to cells to non-CatA expressing cells.
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Latest revision as of 14:41, 27 October 2016

catA-FBD1 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 1 (FBD1, BBa_K2043010) 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-terminus.


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

The gene for the CatA enzyme was taken from Acinetobacter pittii (NCBI Ref. Seq.: YP_004995593.1), which we codon optimized for E. coli with the removal of BsaI restriction sites. A His-tag was also added at the C-terminal end for protein purification.
We wanted to test Catechol-dioxygenases: one was 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 1 (FBD1) has affinity for cotton, linen, polyester, silk and wool. It is positively charged (+1) and it is proline rich. We fused this fabric domain to the N-terminus of catA to improve its ability to degrade anthocyanin-based fabric stains.

Testing the part

We tested the activity of CatA-FBD1 using cell extract of transformants of E. coli overexpressing our protein. We wanted to observe whether the fusion of the FBD1 would affect protein activity or expression. Since the FBD1 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 50 mM at pH 7, with 30 mM of Catechol substrate according to previously established techniques (Lin 2015). Measurements were taken after 35 min, at which time the substrate had been completely consumed by the native protein. Control corresponds to cells 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, at 260 nm. Since much more reaction product is produced with cells expressing CatA than in the control, we can confirm that the enzyme was functional. Fusing the FBD1 retained CatA activity slightly decreased CatA activity.

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