Ma, Liang and Kim, Jungwoo and Hatzenpichler, Roland and Karymov, Mikhail A. and Hubert, Nathaniel and Hanan, Ira M. and Chang, Eugene B. and Ismagilov, Rustem F. (2014) Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s Most Wanted taxa. Proceedings of the National Academy of Sciences of the United States of America . pp. 1-6. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:20140620-103511347
- Published Version
See Usage Policy.
- Supplemental Material
See Usage Policy.
Use this Persistent URL to link to this item: http://resolver.caltech.edu/CaltechAUTHORS:20140620-103511347
This paper describes a microfluidics-based workflow for genetically targeted isolation and cultivation of microorganisms from complex clinical samples. Data sets from high-throughput sequencing suggest the existence of previously unidentified bacterial taxa and functional genes with high biomedical importance. Obtaining isolates of these targets, preferably in pure cultures, is crucial for advancing understanding of microbial genetics and physiology and enabling physical access to microbes for further applications. However, the majority of microbes have not been cultured, due in part to the difficulties of both identifying proper growth conditions and characterizing and isolating each species. We describe a method that enables genetically targeted cultivation of microorganisms through a combination of microfluidics and on- and off-chip assays. This method involves (i) identification of cultivation conditions for microbes using growth substrates available only in small quantities as well as the correction of sampling bias using a “chip wash” technique; and (ii) performing on-chip genetic assays while also preserving live bacterial cells for subsequent scale-up cultivation of desired microbes, by applying recently developed technology to create arrays of individually addressable replica microbial cultures. We validated this targeted approach by cultivating a bacterium, here referred to as isolate microfluidicus 1, from a human cecal biopsy. Isolate microfluidicus 1 is, to our knowledge, the first successful example of targeted cultivation of a microorganism from the high-priority group of the Human Microbiome Project’s “Most Wanted” list, and, to our knowledge, the first cultured representative of a previously unidentified genus of the Ruminococcaceae family.
|Additional Information:||Copyright © 2014 National Academy of Sciences. Freely available online through the PNAS open access option. Edited by Robert Haselkorn, The University of Chicago, Chicago, IL, and approved May 19, 2014 (received for review March 20, 2014) We thank Igor Antoshechkin and the Jacobs Genetics and Genomics Laboratory at California Institute of Technology for help with next-generation sequencing and assembly of the genome. We thank Alasdair McDowall from Prof. Grant Jensen’s lab for assistance with electron microscopy, Whitney Robles for contributions to writing and editing this manuscript, Prof. Tom Schmidt for discussions of 16S rRNA heterogeneity and experimental techniques, and for suggesting the creation of the 100 “Most Wanted” list at the HMP meeting, Prof. Jared Leadbetter for discussion on nomenclature of the isolate, Prof. Victoria Orphan for access to lab facilities, and Dionysios A. Antonopoulos for providing the anaerobic chamber to process the clinical samples. We thank Robert Edgar, J. Gregory Caporaso, Justin Kuczynski, William A. Walters, Hiroyuki Imachi, George M. Garrity, and Ashlee M. Earl for helpful discussions. Research reported in this publication was supported by the National Human Genome Research Institute of the National Institutes of Health under Award R01HG005826. R.H. was supported via an Erwin Schrӧdinger Postdoctoral Fellowship by the Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung; J 3162-B20). Author contributions: L.M., J.K., R.H., E.B.C., and R.F.I. designed research; L.M., J.K., R.H., M.A.K., N.H., and I.M.H. performed research; L.M., J.K., and R.H. analyzed data; and L.M., R.H., and R.F.I. wrote the paper. Conflict of interest statement: R.F.I. has a financial interest in SlipChip Corporation. This article is a PNAS Direct Submission. Data deposition: The genome sequences reported in this paper have been deposited in the Joint Genome Institute’s Integrated Microbial Genomes database: https://img.jgi.doe.gov/cgi-bin/w/main.cgi (accession no. 2545555870). The 16S rRNA gene sequences of isolate microfluidicus 1 reported in this paper have been deposited in the GenBank database (accession nos. KJ875866 and KJ875867). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1404753111/-/DCSupplemental.|
|Subject Keywords:||microscale; anaerobe; aerobe; cultivate; metagenome|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||George Porter|
|Deposited On:||25 Jun 2014 20:41|
|Last Modified:||25 Jun 2014 22:01|
Repository Staff Only: item control page