Difference between revisions of "Part:BBa K2043002"

Revision as of 01:54, 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. Such enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol. EC number is 1.13.11.1. The fabric domain was fused in N-terminal.

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

CatA enzyme originally comes from Acinetobacter pittii (NCBI Ref. Seq.: YP_004995593.1), which we codon optimized for E. Coli avoiding 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 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 charget (+1) and it is proline rich. We fused a fabric domain with catA hoping to improve its function as stain removal.

Testing the part

We tested the activity of CatA-FBD1 using cell extract from transformants of E. coli overexpressing our protein. We wanted to observe whether the fusion of the FBD1 would affect the activity, or the expression of the protein. 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 2CatA fusion proteins' activity was measured in Sodium Phosphate 50mM at pH 7, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 35 min, timepoint at which all the substrate had been consumed by the native protein. Control corresponds to cells that do not express our proteins.

As the image indicates, there is a clear difference between our native and fusion enzymes and the control. We measured the reaction product, cis,cis-muconic, at 260nm. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional. However fusing 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

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]

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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 1 (FBD1, <a href="https://parts.igem.org/Part:BBa_K2043010">BBa_K2043010</a>) cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. Such enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol. EC number is 1.13.11.1. The fabric domain was fused in N-terminal.
+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 1 (FBD1, <a href="https://parts.igem.org/Part:BBa_K2043010">BBa_K2043010</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>. Such enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol. EC number is 1.13.11.1. The fabric domain was fused in N-terminal.
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