Related Experiment Video
Updated: Sep 30, 2026

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells
Published on: March 3, 2015
Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase
Diane Barriault1, Marie-Michèle Plante, Michel Sylvestre
1Institut National de la Recherche Scientifique INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, Canada.
Abstract:
In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of bphA that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of bphA genes from Burkholderia sp. strain LB400, Comamonas testosteroni B-356, and Rhodococcus globerulus P6. Unlike all parents, these variants exhibited high activity toward 2,2'-, 3,3'-, and 4,4'-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (335TFNNIRI341) of LB400 BphA by the corresponding segment (333GINTIRT339) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.
Related Concept Videos
Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
Hydrolysis of Chlorobenzene to Phenol: Dow Process
Regioselectivity and Stereochemistry of Hydroboration
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
[3,3] Sigmatropic Rearrangement of 1,5-Dienes: Cope Rearrangement
Benzene to 1,4-Cyclohexadiene: Birch Reduction Mechanism
Reactions of Aldehydes and Ketones: Baeyer–Villiger Oxidation
The carbonyl center is activated by...

