Development of simple and rapid diagnostic system using genetically engineered mutant Escherichia coli

Abstract

A cell-based quantitative assay system for Hcy, utilizing two Escherichia coli auxotrophs that are genetically engineered to grow in the presence of methionine (Met) and either homocysteine (Hcy) or Met, has been developed. A bioluminescent reporter gene, which produces luminescence as cells grow, was inserted into the auxotrophs, so that cell growth can be readily determined. By measuring relative luminescence unit (RLU) values from the two auxotrophs immobilized within agarose gels arrayed on a well plate, the amount of Hcy is quantitatively determined based on differences between two RLU values corresponding to cell growth of two auxotrophs with excellent levels of precision and reproducibility. Finally, the diagnostic utility of this assay system was verified by its employment in reliably determining different stages of hyperhomocysteinemia in human plasma samples. In contrast to existing conventional methods, the new system developed in this effort is simple, rapid and cost effective. As a result, it has great potential to serve as a viable alternative for Hcy quantification in the diagnosis of hyperhomocysteinemia. We have developed a new cell-based galactose assay utilizing two bioluminescent Escherichia coli immobilized within agarose gel arrayed on a well plate. galT knockout strain (galT(-) cell) was genetically engineered to inhibit the cell growth in the presence of galactose in a sample, thus its growth was proportional to glucose concentration. On the other hand, E. coli W (normal cell), which is original strain before the genetic engineering to obtain galT(-) cell, normally grew in the presence of either galactose or glucose. Into the two E. coli strains, a bioluminescent reporter gene which produces luminescence as cells grow was transformed for facile measurement of the cell growth. Therefore, a relative luminescence unit (RLU) value corresponding to the galT(-) cell growth induced by glucose was subtracted from the RLU value corresponding...