Development of an Optimized Medium,Strain and High-Throughput Culturing Methods for Methylobacterium extorquens
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Authors: | Nigel F. Delaney Maria E. Kaczmarek Lewis M. Ward Paige K. Swanson Ming-Chun Lee Christopher J. Marx |
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Affiliation: | 1. Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, United States of America.; 2. Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, Massachusetts, United States of America.; Iowa State University, United States of America, |
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Abstract: | Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new MethylobacteriumPIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens. |
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