Difference between revisions of "Part:BBa K2043003"

Revision as of 15:28, 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 also added to 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 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-muronic 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.

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.

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 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>. 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 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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@@ Line 11: / Line 11: @@
 <b>Figure 1</b> Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.<br><br>
-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 also added at the C-terminal end for protein purification.
+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 also added to the C-terminal end for protein purification.
 <br>
 We wanted to test Catechol-dioxygenases: one was 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>
-<b>The Fabric Binding Domain 10 (FBD10)</b> has affinity for cellulose and has a positve charged (+1). We fused a fabric domain to the N-terminus of CatA hoping to improve its ability to bind to and degrade fabric stains.
+<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.
 <br>
 <br>
@@ Line 29: / Line 29: @@
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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, cis,cis-muconic, at 260nm. Since much more reaction product is produced 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.
+We observed a clear difference between our native and fusion enzymes and the control (Figure 2). We measured the reaction product, cis,cis-muronic 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.
 <br><br>