The quest to characterize the human microbiome is a daunting goal, but one that promises to enhance significantly our understanding of health and our management of a wide variety of disease states. In this quest, two features of the human microbiota in particular, pose major challenges: the large proportion and number of as-yet uncultivated species, and the extreme unevenness of the microbial communities, with a resulting large number of potentially important community members that fail to be """"""""seen"""""""" in routine surveys. The ability to identify, isolate, and sequence the genome of single bacterial cells would allow us to characterize and understand both rare and uncultivated microbial species, and materially advance our understanding of the human microbiome. In recent work, a microfluidic device has been designed and fabricated, with features that mimic an integrated electrical circuit;this device isolates individual bacterial cells, and allows their genome to be amplified in nanoliter volumes. In this Application, a plan is proposed for optimization and augmentation of this microfluidics device, so that environmental contamination is reduced, rare cell types are more easily captured, larger numbers of cells are screened more quickly, and gene expression is more easily measured from single cells. The long-term objectives of this work are to enhance our understanding of the human microbial communities, and in particular, of novel or poorly-characterized, uncultivated microbial community members. This proposal responds to critical unmet needs posed by the NIH Human Microbiome Project. The following are the Specific Aims of this proposal:
Aim 1. To reduce the contribution of environmental DNA to single cell genomic sequence data, and increase the """"""""signal-to-noise"""""""" ratio of the sequence data obtained with our cell- sorting, genome amplification microfluidics device. The experimental approach involves the integration of optical (laser) tweezers into the device.
Aim 2. To improve the ability to detect and capture rare microbial community members with the microfluidics device. The experimental approach involves the integration of fluorescence in situ hybridization techniques, specific probes, and fluorescence imaging with the microfluidics device.
Aim 3. To increase the speed of single cell selection and isolation with the microfluidics device. The experimental approach involves more highly parallel microdevice designs, optimization of laser power and laser optical path, and further automation of cell manipulations.
Aim 4. To enhance the capability for gene expression analysis in single bacterial cells. The experimental approach involves the development of on-chip protocols for RNA isolation, reverse transcription, and use of digital PCR to quantify transcript abundance from single cells.
This research is relevant to public health because it will lead to a better understanding of the microorganisms that live on and in the human body. Because the human indigenous microbial communities help to maintain health and when disturbed, contribute to disease, this research may lead to new tests for diagnosing or predicting disease associated with microbial community disturbance, and new strategies for maintaining or restoring health.
