Optimizing Growth Conditions for M. extorquens

We first tested whether we could reliably measure M. extorquens exponential growth rates using a robotic measurement system under similar conditions to those used by previous investigators with E. coli[3] (link). AM1 cultures were grown in a Microtest 96-well tissue culture treated plates (Falcon-35-3072) using buffered medium comprised of 14.5 mM of K₂HPO₄, 18.8 mM of NaH₂PO₄, 8 mM ammonium sulfate, 20 µM calcium chloride and the C7 metal mix that was left unchelated (i.e., no citrate) with 17 mM methylamine·HCL added to the base medium. The mixture was aliquoted in 160 µL portions into wells of a 96-well plate. The growth curves from the initial tests in 96 well plates showed huge deviations from the exponential model and were exceptionally noisy. We concluded that 96-well plates were inadequate for sustained exponential growth of M. extorquens. We re-evaluated this conclusion at the end of this project, after optimizing our strains and media, by again growing M. extorquens in MP media and in 96-well plates (Fig S1). Although exponential growth could then be achieved, relative to a 48-well plate grown simultaneously with the same inoculum, the average estimated growth rate was 7% slower and the std. error was 50% larger.
In contrast, we found that 48-well plates did allow for adequate mixing and consistent exponential growth (Fig 1D). We therefore altered the robotic system by installing new custom-built racks so that it could use 48-well plates (CoStar-3548) instead of 96-well plates. In contrast to the 96-well plates where the medium did not appear to move within the well, the media in the 48-well plates rhythmically swirled around. We also tested a second type of 48-well plate, from the Cellstar line made by Greiner Bio-One (Catalog #677 102). Surprisingly, although medium in the CoStar plates visibly swirled while shaking, the meniscus in the Cellstar plates, as in the 96-well plates, stayed at approximately the same level and did not appear to move; correspondingly, cultures grew much more poorly in Cellstar plates. For all future work in this paper, we grew the cultures in CoStar plates in 640 µL per well with the incubator shaking at 650 RPM, as growth and the swirling of the liquid appeared to be as good or better than the range of other values tested.