Researches On The Colorable Marker System Of XylE Gene-Catechol 2, 3-dioxygenase And Its Application To The Study Of Genetic Engineering

Catechol 2,3-dioxygenase (CatO₂ase) is encoded by xyIE gene. Funtional expression of the gene was detected by spraying antibiotic selection plates with an aqueous solution of 0.2-0.5 M catechol. Colonies of bacterium cells that express the gene become yellow due to the conversion by CatO₂ase of Catechol to 2-bydroxymuconic semialdehyde (HMS), The system described here offers several advantages: (1) Specially prepared indicator plates are not necessory to monitor xylE gene expression; (2) The CatO₂ase substrate, Catechol is very cheap. (3)The catechol spray test is very simple, sensitive and rapid; (4) CatO₂ase specific activities are determined by a simple spectro-photometric assay.A series of new plasmids containing xylE gene was constructed based on the shuttle plasmid pTG402 between E.coli and B.subtilis. The expressed xylE gene product CatO₂ase was meassured for its output and distribution, and analysed for its structural hydropbobicity and hyarophilicity, It was found that the output of CatO₂ase was relative to plasmids, host ceils, culture time and with or without induction.