The field of microbiology has been advanced through the use of new technologies. In recent years, PCR and high throughput DNA sequencing have rapidly propelled microbiology away from its historical dependence on cultivability and morphological classification. However, despite the relative ease with which whole genomes are being obtained, high throughput technologies have not significantly improved our ability to cultivate novel microorganisms in the laboratory. It is well known that many microbes function in syntrophy, exchanging metabolites frequently to sustain their own metabolism. This work proposes that significant enhancements can be made to understanding of microbes, member by member, by developing technologies to foster their syntrophic growth while maintaining their spatial segregation. These issues will be addressed by creating microfluidic cell culture systems with porous walls that allow bacteria to communicate chemically while being physically isolated. The proposed systems will be fabricated using a novel implementation of the well established process of electrophoretic deposition (EPD). The fabrication technique developed in this research will have high impact in microbiology, as well as the micro electromechanical systems (MEMS) and microfluidics communities. This development will transform the way cultivation of previously uncultivable bacteria and archaea is approached, and be a watershed for microbiological research. The principal investigators will leverage several existing partnerships to impact the educational environments at MIT and Harvard. Examples include student mentoring through the MIT Summer Research Program (MSRP), incorporating EPD into a laboratory course at MIT, and training of undergraduates in the Girguis laboratory through the Howard Hughes Medical Institute Undergraduate Research Fellowship (HHMIUF) program.
?? The field of microbiology has been advanced through the use of new technologies. In recent years, PCR and high throughput DNA sequencing have rapidly propelled microbiology away from its historical dependence on cultivability and morphological classification. However, despite the relative ease with which whole genomes are being obtained, high throughput technologies have not significantly improved our ability to cultivate novel microorganisms in the laboratory. It is well known that many microbes function in syntrophy, exchanging metabolites frequently to sustain their own metabolism. Many believe that one of the reasons more than 99% of all bacteria on the planet have never been isolated or cultured is that we lack the appropriate cultivation platforms. To date, the state of the art technology in engineered cellular micro-environments has been the use of droplets to segregate cells. However, this technology does not allow for chemical diffusion or transport between individual droplets, making it unsuitable for the growth of syntrophic communities (the thrust of this work). Intellectual Merit We have addressed these issues by successfully creating microfluidic cell culture systems with porous walls that allow bacteria to communicate chemically while being physically isolated. The porous walls allow chemicals to move between culture chambers but prevent microbe cross contamination. If the chambers are filled with one microbial cell, the porous walls ensure pure cultures which can be used for genetic analysis after cultivation. Further, our cell culture system features very small volumes (1 nanoliter) and we've shown that it's biocompatible by growing several strains of Escherichia coli. Broader Impacts This work broadens the horizon of what's possible for microbiologists studying bacteria cultivation and community dynamics. For example, with our system it is now possible culture cells (all types of cells, not just bacteria) in a low stress environment that allows for physical isolation. Here we focused on difficult to culture microbes, but future work could explore diverse areas such as quorum sensing and microbial biosynthesis. This work also advances the fields of microfluidics and microfabrication by showing that it's possible to create microscale porous features with good structural integrity in a high throughput manner.
