There is growing interest in rapid microbial pre-concentration methods to lower the detection limit of bacterial pathogens of low abundance in samples. Here, we report an integrated microfluidic PCR system that enables bacterial cells of interest in samples to be concentrated prior to PCR. It consists of two major compartments: a preconcentration chamber for the immunomagnetic separation of bacterial cells, and a PCR chamber for the DNA amplification of the concentrated cells. We demonstrate the feasibility of the system for the detection of microbial pathogens by preconcentrating the human pathogen Escherichia coli O157:H7, and also amplifying its DNA. The detection limit of E. coli O157:H7 in the PCR system is 1 × 10 CFU (colony forming unit)/mL. On-chip processing steps, including preconcentration and PCR steps, take less than two hours. Our system can serve as a rapid, specific, and quantitative platform for the detection of microbial pathogens in samples of large volume.
In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.
A ZraP-based lead sensing and removal system was constructed in E. coli. It was regulated by the ZraS/ZraR two-component system. The expression profile of the zraP gene towards extracellular lead was studied via real-time PCR. A dual-function bacterial system was also designed to express GFP and OmpC-lead binding peptide under the control of zraP for the simultaneous sensing and adsorption of environmental lead without additional manipulation. The constructed bacterial system can emit fluorescence and it adsorbed a maximum of 487 µmol lead/g cell DCW. From a study of artificial wastewater, the constructed bacteria adsorbed lead highly selectively (427 µmol lead/g cell DCW) among other metal ions. The newly-constructed dual function bacterial system can be applied for the development of an efficient process for the removal of lead from polluted wastes.
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