Connolly, James M.Kaufman, M.Rothman, Adam P.Gupta, R.Redden, George D.Schuster, M.Colwell, F.Gerlach, Robin2017-01-272017-01-272013-09Connolly J, Kaufman M, Rothman A, Gupta R, Redden G, Schuster M, Colwell F, Gerlach R, "Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation," Journal of Microbiological Methods, September 2013 94(3): 290–299.10.1016/j.mimet.2013.06.028https://scholarworks.montana.edu/handle/1/12453Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.Article