The fermented probiotic milk beverage, kefir, is made by grains consisting of an consortium of microbes (including lactic acid bacteria and yeasts) surrounded by a exopolysaccharide matrix. The members of this consortium have not been fully characterized by culture-independent methodologies, nor has the population dynamics of these kefir grains been determined.
Using culture independent methodology, we propose to investigate the population structure and possible dynamics of this microbiological consortium under several conditions: from isolate to isolate locally, from isolates at different geographical locations, and before and after changes in cultivation conditions (moving from milk to grape juice or sugar-water). This approach should reveal the level of population diversity among the residents of active fermenting grains, and how the population structure is altered by changing culture conditions.
In addition, we propose to investigate if �non-kefir associated� lactic acid bacteria can become incorporated into kefir grains during culture, using GFP labeling to reveal �microgeographic� aspects of this process, if it occurs.
Finally, we will develop a �virtual meeting place� for investigators interested in this phenomenon: �Kamp Kefir.� This site will contain sequence data from experiments such as we describe carried out by as many investigators as have interest in this approach, as well as information about kefir and data pertaining to this interesting system. The resident data will serve as an excellent teaching tool for students, particularly students interested in bioinformatics, metagenomics, and related technologies, at institutions that do not have active research programs.
Standard microbiological equipment and supplies, kefir grains from various sources, gfp-shuttle vector, universal 16s and 18s rRNA primers, PCR reagents and equipment, TA-TOPO cloning kit, competent E. coli cells, plasmid miniprep supplies, access to DNA sequencing facilitity.