Developing a low-cost milliliter-scale chemostat array for precise control of cellular growth

Abstract

Multiplexed milliliter-scale chemostats are useful for measuring cell physiology under various degrees of nutrient limitation and for experimental evolution. In each chemostat, fresh medium containing a growth rate-limiting metabolite is pumped into the culturing chamber at a constant rate, while culture effluent exits at an equal rate. Although such devices have been developed by various labs, key parameters -the accuracy and precision of flow rate and the operational rangeare not explicitly characterized. Here we report the development of multiplexed milliliter-scale chemostats where flow rates for eight chambers can be independently controlled to vary within a wide range, corresponding to population doubling times of 3~ 13 hours. Importantly, flow rates are precise and accurate without the use of expensive feedback systems. Among the eight chambers, the maximal coefficient of variation in flow rate is less than 3%, and average flow rates are only slightly below targets, i.e., 3-6% for 13-hour and 0.6-1.0% for 3-hour doubling times. This deficit is largely due to evaporation and should be correctable. We experimentally demonstrate that our device allows accurate and precise quantification of population phenotypes.

Uncovering and resolving challenges of quantitative modeling in a simplified community of interacting cells

“…In chemostat measurements, we can control the environment to mimic those encountered by the community. However, in addition to the time-consuming nature of constructing and calibrating chemostats to ensure accurate flow rates [58], rapid evolution occurs. For L − A + , mutants pre-exist at a low frequency but can grow severalfold faster than the ancestor (S7 Fig).…”

“…We have constructed an eight-vessel chemostat with a design modified from [75]. For details of construction, modification, calibration, and operation, see [58].…”

Metabolic excretion associated with nutrient–growth dysregulation promotes the rapid evolution of an overt metabolic defect

“…When in chemostat mode, a constant average flow rate f of medium into the vessel was maintained. Unlike our earlier chemostat setup [ 77 ], here, constant flow rate was achieved via a scale (Ohaus SPX2201) that constantly weighed its associated reservoir, and the reading was acquired through an RS232 interface (Ohaus Scout RS232). The initial scale reading, M initial , was recorded when the chemostat channel was activated or reset.…”

“…Second, regardless of whether live cells were defined as fluorescent-protein–expressing cells, colony-forming cells, or cells with intact membrane and thus capable of excluding the nucleic acid dye ToPro3, we obtained similar quantification of live-cell densities. Specifically, adenine-auxotrophic (A) and lysine-auxotrophic (B) cells expressing fluorescent proteins were grown in adenine-limiting and lysine-limiting chemostats, respectively [ 30 , 77 ]. After cell density had reached a steady state, a sample was taken in which a portion was plated on rich medium, and live-cell density was subsequently quantified from colony counts.…”

Disentangling strictly self-serving mutations from win-win mutations in a mutualistic microbial community

“…We have constructed an eight-vessel chemostat with a design modified from Takahashi et al (2015). For details of construction, modification, calibration, and operation, see Skelding et al (2017). A detailed discussion on using chemostats to quantify release and utilization phenotypes can be found in Hart et al (2019a).…”