Continuous flow systems

As we detailed above, bacteria grow quickly and the need for fresh nutrient is a lim- iting factor in most biological applications. If the right molecules do not surround cells or if these molecules are in insufficient quantities, then bacteria slow down their growth in what is called a stationary phase (cf. Figure 3.11)[233]. Although it is always possible to redilute stationary cultures into fresh medium, bacterial growth is physi- ologically affected by periods of starvation (maximal cell density in the medium, no more growth)[158,264]. One solution to this problem is to cultivate cells in continuous growth conditions via bioreactors. Bioreactors can be assembled in order to provide a constant supply of fresh nutrients to growing cultures and, therefore, allow a better tracking of growth experiments.

Bioreactors require a deeper understanding of cell growth as represented in Fig- ure 3.11[262,107]. In Figure 3.11, phase 3 shows the steepest increment in cell density over time. Derived by plotting optical density measures at λ = 600nm over time, the exponential growth phase can be used to approximate cellular division rate[158, 147]. Knowledge of the frequency of cellular division (coupled with replication of one copy of the chromosome) is essential for the work with bioreactors. Chemostats, a specific type of bioreactor, are designed to have a continuous flow system which constantly provides fresh medium to an ongoing bacterial culture. To run this system, a manifold ensures that excess medium is extracted from growing cultures, and this volume must be equivalent to the fresh nutrient input rate[208, 226]. As a whole, chemostats allow

FIGURE 3.11: Bacteria undergo 6 consecutive phases during growth:

(1) the lag phase (null growth), (2) growth acceleration, (3) exponential growth, (4) late-exponential growth (or retardation phase), (5) stationary phase (or no more growth) and (6) biological decay. Error bars show stan-

3.3. Large-scale screening setup 65 cultures to be grown at a constant rate that can be controlled via a pump settings, where the best rate is equivalent to bacteria growth rate measured in exponential phase. With the approximation of a culture growth rate, it is relatively straightforward to test and derive an appropriate chemostat dilution rate to maintain constant growth with a suf- ficient nutrient input, and to sustain near-optimal growth conditions for long periods of time.

Although mechanistic aspects of chemostats are well-defined, a thorough calibra- tion process is necessary to obtain relevant data. As shown in Figure 3.12 (assembly), tubing first needs to be cut and assembled with connectors in order to build the man- ifold[3, 216]. In our setup, four 50ml vessels could be followed simultaneously and it was important to match all tubing dimensions in order to get an identical flow rate across multiple samples. Calibration could be carried out with water, since LB is water- based and both substances share similar viscosity, and was repeated after instruments sterilisation at least twice to make sure culture dilution rates remained stable. Once the chemostat calibration was verified and validated, all autoclavable units were sterilised and prepared for assembly immediately after autoclaving, as shown in Figure 3.12.

For this study, the benefit of using a chemostat was to obtain accurate predictions of the number of bacterial replication events and thus information regarding barcode replication cycles. Given a bacterium growth rate, it is easy to program a calibrated chemostat with settings that would comply with these specific growth requirements. Therefore, the number of replication cycles can be precisely obtained by actively dilut- ing growing cultures at a specific rate. However, cells first need to reach exponential growth, and the number of generations between a culture inoculation and exponen- tial growth is usually a rough estimate. As a standard method in microbiology, one can estimate the number of viable bacteria inoculated onto a plate via the number of colony forming units (CFUs) obtained after overnight growth. For the purpose of this study, we performed CFU growth curves and serially diluted cultures over time to obtain ideal dilution rates at which a minimal number of cells could be used to inoc- ulate a chemostat. Given this minimum amount of cells, it was possible to evaluate, given a specific growth rate, the time needed for cultures to attain exponential phase (e.g. when the chemostat continuous flow should be turned on). Although chemostat calibration defined the exact dilution rate of growing cultures, it always took between 3 and 5 vessel volumes of fresh medium for bacteria to reach steady-state growth. Therefore, along all chemostat experiments, we recorded optical density (OD) mea- surements while sampling cells for barcode sequencing to ensure estimated dilution rates were accurate and cultures could reach steady-state growth.

FIGURE 3.12: The setup panel (top left) outlines the tubing necessary to

connect the multiple components of a chemostat with their associated plugs (male/female luers and syringes), without forgetting a magnetic stirrer to keep continuous cultures well-mixed. Autoclaving (top right panel) must be performed on all sterilisable equipment in an organised manner, since chemostats should be started as soon as their equipment has been autoclaved. Calibration (bottom panel) can be performed with water and should be timed and measured to match bacteria optimal growth rate.

3.3. Large-scale screening setup 67 F IG U R E 3 .1 3 : (A) shows simplified schematics of the chemostat manifold and components. (B) top-left pictur e shows the actual setup featuring 4 individual replicates. (B) right hand side pictur e is a zoomed in image of cultur e vessels, allowing to see dif fer ential needles level while (B) bottom left pictur e is a top view of the chem ostat manifold

A details schematics of the continuous flow system: under a constant vacuum, fresh medium is pumped from a reservoir via a peristaltic pump to provide nutrients to an ongoing bacterial culture. Air pressure allows to keep the cultures dilution with fresh nutrients balanced with waste removal. This is done via a custom fusion of an Akta head to a Duran bottle cap, photographed in Figure 3.13B on the right. This picture allows to see three 14G serological needles set at different levels to adjust the bacterial culture volume with input, output and pressure ports. These caps were used to seal culture vessels in combination with a circular piece of silicon to act as a joint. We also coated silicon with vaseline to keep vessels sealed after autoclaving and to ease setup of the chemostat manifold. Pressure problems often come as the main issue in the setup of chemostats and setting up four individual vessels per experiment always allowed us to keep at least three of them under appropriate experimental conditions.

Here, we detailed the system through which we studied the replication of DNA barcodes over an exact, large number of bacterial generations. However, the experi- mental conditions of these assays do not represent general growth methods that are usually undertaken when growing bacteria. Therefore, we also studied DNA barcodes via a "laboratory-like" fashion, where cells would be grown and rediluted for an ex- tensive number of times between freeze/thaw cycles. The next paragraph details the setup of this assay.