Difference between revisions of "Part:BBa K2043007"

Revision as of 11:13, 24 October 2016

bpuI laccase codon optimized for E. coli

This part corresponds to Bacillus pumilus laccase cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from Bacillus pumilus, 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.

This gene had already been registered in the Biobrick Registry, in part BBa_K863000.

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, laccases are good candidates because they are known to degrade Anthocyanins.

Testing the part

We tested the activity of BpuI using cell extract of cells expressing our protein.
First, we performed an SDS-PAGE to check whether the protein was being expressed.

The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity.
We tested our cell extract for BpuI activity in Citrate Phosphate Buffer at pH 4, with 0.4mM of ABTS being used 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.

As the image indicates, there is a clear difference between our enzyme and the control. We measured the reaction product at 420nm, which results from the oxidation of ABTS. Since much more reaction product is produced with cells expressing CatA than in the control, we can affirm that the enzyme was functional.

Sequence and Features

Cho, E. A., Seo, J., Lee, D. W., & Pan, J. G. (2011). Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores. Enzyme and microbial technology, 49(1), 100-104.

Reiss, R., Ihssen, J., & Thöny-Meyer, L. (2011). Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC biotechnology, 11(1), 1.