Abstract
A single-step plate method is described for the screening of transformants of cloned gene xyl-A encoding xylose isomerase. It is rapid, inexpensive, easy to perform, and more specific, as the selection is based on xyl-A expression level. The crucial composition of the plate medium comprised agar, buffer, nutrients, co-factor, substrate, inducer, antibiotic, and an indicator, i.e., 2, 3, 5-triphenyl tetrazolium chloride (TTC). Transformed cells of E. coli BL21 (DE3) with a plasmid (pET21(a)++xyl-A), were exclusively capable of growing, isomerizing the substrate, and producing deep-red formazan by reducing TTC, whereas the untransformed cells and cells containing a plasmid without an inserted gene failed to grow. Thereby, it deprived the need for a blue/white screening assay. TTC reduction is essentially irreversible; hence, the color remained stable. TTC is readily metabolized in the respiratory chain and does not affect the viability of microorganisms; therefore; there is no need for replica plating. Hence, it appeared to be an appropriate method for screening xyl-A mutant libraries. It will create a paradigm for future studies on the evolution of the xyl-A gene.
Article History
Received: Nov 02, 2024; Accepted: Aug 14, 2026; Published: Sep 30, 2026
Recommended Citation
Fatima, B.,
& Javed, M. M.
(2026).
A Single-Step Plate Method for the Screening of Xyl-A Mutant Libraries Based on Gene Expression,
Journal of Bioresource Management, 13
(3).
