Medium shift and determination of lag times

Exponentially growing cultures in preshift condition were obtained following the protocol outlined above for growth measurements in tubes or flasks for metabolomics and proteomics experiments. Cultures were grown up to OD₆₀₀~0.5 before the shift was performed. Cells were then carefully transferred to a filter (previously washed with Milli-Q water) to remove preshift medium and washed twice with warmed postshift medium (at least twofold the volume of culture transferred to the filter). The filter was then moved to a sterile 50ml Greiner tube with warmed postshift medium and cells were gently resuspended from the filter by pipetting. Cells were then diluted in warmed postshift medium to OD₆₀₀~0.05 for the purpose of lag time measurements and to OD₆₀₀~0.5 for the purpose of metabolomics and proteomics measurements and incubated. The entire shift was typically completed in under 5 minutes. Lag times were determined as follows: After cells reached steady-state growth in postshift condition, about three to four OD₆₀₀ data points were fitted with an exponential function. The intersection of the fitted exponential and initial postshift OD₆₀₀ was used to determine the lag time.

To screen combinations of carbon sources using a plate reader, the protocol was slightly modified. After being transferred to the filter, cells were washed twice and resuspended using warmed medium without a carbon source. Cells were then diluted into the pre-warmed Thermo Fisher Scientific Nuclon 96 well bottom flat transparent plates filled with different postshift media. These plates covered with a lid were then incubated and culture density was monitored using a Tecan Infinite M200 plate reader at 37°C shaking at 880 rpm to measure lag times. Lag times were determined by fitting the growth curve over the range, where maximal exponential growth rate was reached, by the function OD(t) = OD_initexp[λ(t−T_lag)], which is an exponential growth curve with growth rate λ that is shifted by the lag time T_lag. OD_init is the OD₆₀₀, measured just after the shift and the fit parameters were the growth rate growth rate λ and the lag time T_lag. The fit was performed using the ‘fit’ command of Gnuplot, which is an implementation of the nonlinear least-squares (NLLS) Marquardt-Levenberg algorithm.

A single colony of B. subtilis 3610 was inoculated in 3 ml LB in the morning as a seed culture at 37°C. In the evening, the seed culture was diluted into minimal medium containing various carbon sources: 20 mM glucose, 20 mM mannose, 20 mM maltose and 40 mM glycerol to ensure exponential growth the next day. The seed culture was then diluted to an OD₆₀₀ of 0.025. When the culture reached an OD₆₀₀ of 0.2–0.3, the cells were centrifuged, washed with prewarmed postshift medium, and shifted to minimal medium containing 60 mM Acetate. The OD values were recorded by BioTek Synergy H1 microplate reader.

Overnight seed cultures of S. cerevisiae YPS128 and YPS163 were grown in chemically defined synthetic complete media^39–41, containing 2 % (w/v) glucose. The next day, the seed culture was diluted to an OD₆₀₀ of 0.025 in synthetic complete medium containing various single carbon sources: 2 % (w/v) of glucose, galactose, maltose or raffinose and incubated at 30°C. When cultures reached the exponential phase (OD₆₀₀ of 0.2–0.4), cells were washed twice with prewarmed postshift medium and shifted to the postshift medium containing 2% (w/v) acetate. Growth was followed and OD₆₀₀ values were recorded in a BioTek Synergy H1 microplate reader. The chemically defined synthetic complete media used for this yeast carbon switch experiment left out inositol completely to ensure that cells were only growing one a single carbon source.

We use a microfluidic platform based on the ‘mother machine’ design⁴², to track individual cells during lag phase. We monitor the morphology of individual cells as they experience media switch under controlled conditions and use the morphological measurements to obtain both growth rate and lag times of individual cells (single-cell lag time analysis).

The mother machine microfluidic device, where cells grow and divide in narrow trenches and are fed by diffusion by an orthogonal feeding channel, has been used for long term tracking of cells^42,43 under tightly controlled local conditions. The Paulsson lab has recently developed a microfluidic platform for tracking cell lineages along the growth curve (Bakshi, S., Leoncini, E., Baker, C., Cañas-Duarte, S., Okumus, B. and Paulsson, J., bioRxiv 2020.03.27.006403), where a batch culture is connected to a microfluidic chip. We use this platform to obtain lag time information at single-cell level (see Extended Data Fig. 3a). Cells from YCE44 strain (expressing constitutively mCherry1–11-mKate) were loaded in a mother machine chip and were allowed to recover for several hours in N+C+ glucose minimal medium before starting imaging. A flask with glucose medium inoculated with YCE44 strain was then connected to the microfluidic device, so that the cells in the chip share the same environment as the cells in the flask. The platform enables us to monitor the OD of the batch culture at high frequency (30 seconds), and to grow the culture under usual laboratory conditions (37°C on orbital shaker, 220rpm). This allows us to monitor the behavior of the batch culture and individual cells synchronously. To perform the shift to acetate, cells in the flask were washed twice with postshift acetate minimal medium and resuspended in postshift acetate minimal medium as described in the batch protocol above. After the shift, cells kept growing for some time at the same growth rate both in the flask and in the microfluidic chip, possibly because some glucose medium was still present in the system. After about 60 minutes, glucose ran out and the cells underwent a diauxic shift. We kept monitoring cells in the Mother machine over the course of the lag phase, as they responded to changes in the batch culture. The experimental protocol is illustrated in Extended Data Fig. 3b.

Conditions of cells in the microfluidic chip are not identical to the ones in the flask since, for instance, cells under observation are diffusely fed in the growth trenches. We minimized this effect by using shorter growth trenches (20μm in length). Also, in order to reduce mixing of glucose and acetate media at the time of switch, we introduced a waste line before the microfluidic chip, which allows to divert the flow at the time of switching, to better control the switch dynamics for the cells in the mother machine.

Images were acquired using a Nikon Ti inverted microscope equipped with a temperature-controlled incubator (OKO lab), an Andor Zyla 4.2 camera, a 40x Phase 2 Plan Apo (numerical aperture NA 0.95, Nikon), an automated motorized stage (Nikon) and Lumencor SpectraX light source. All images were acquired with a 1.5x post-magnification, and the camera-objective combination gave a 0.11 μm/pixel. Focal drift was controlled by the Nikon Perfect Focus System. The timelapse imaging and automatic stage movements were controlled by Nikon NIS Elements software. We imaged cells in phase contrast and RFP channel. Images were taken every 15 minutes with 200ms exposure in order to reduce photobleaching and phototoxicity.