Considerations for your first eVOLVER experiment

Just wanted to list out a couple of considerations to think about when transferring your batch culture experiments over to eVOLVER for the first time.

Growth Conditions:
eVOLVER can control temperature, stir, media in/out, and cell density, in 16 independent vials, depending on what control script you write (Python). You can also hook up a different media to each vial. Below are some parameter ranges that are easy to achieve for your first expt, though with some tinkering in future expts you may be able to push outside this range.

Temperature: +3C to +20C above ambient
Stir: up to 1500 rpm
Fluidics: up to 1mL/s flow rates (for peristaltic pumps)
OD: up to OD 0.8, to an accuracy of +/- 0.01, though most commonly we work around 0.1 - 0.4.

The Most Common Experiments:
Batch growth curve:
No fluidics, just monitor 16 different batch cultures tracking optical density every ~20s. Good for grow/no grow experiments, measuring growth rate in different temps/medias with easy setup/less media usage.

Continuous culture where the flow of media in/out is held constant (i.e. a dilution rate of 0.25 hr-1 meaning F/V=0.25 where F is the pump flow rate in mL/h and V is the culture volume in mL), and cells grow up to a steady state where growth rate = dilution rate. Densities will be higher in more permissive conditions (or slower dilution rates), and lower in harsher conditions (or faster dilution rates). Good for keeping growth conditions constant (esp. for modeling), but real-time data is less interpretable. Selects for efficient growth.

Continuous culture where you set an upper and lower density threshold. When cultures grow up to the upper threshold (say OD600 = 0.4) a dilution is triggered to bring down to the desired lower threshold (say 0.2). Sawtooth traces are generated, so can fit an exponential curve on each to calculate growth rate in real time. Good for watching cultures speed up or slow down in real time & no risk of diluting all cells out of culture, but harder to model as conditions slightly change. Selects for fast growth.

Media/Innoculum Requirements:
Continuous culture requires a lot of media, as cells are kept at lower densities than in batch culture, and the culture volume are on the order of 20 or 25 mL. This may become expensive if you use lots of expensive reagents. Running out of media will affect your results, so having extra media is good. For a chemostat, you can exactly calculate how much media you will use, i.e. 0.25 hr-1 dilution rate would use 25mL*0.25 = 6.25 mL per hour. In turbidostat you can calculate how much is used with the following equation:

volume* ln⁡((upper thresh)/(lower thresh)) = media used per dilution

So for a 25mL culture held between OD 0.2 and 0.4, with a 2 hour doubling time, it’ll use ~17mL per dilution, so 8.5 mL/hr. So for a 5 day experiment, each vial would require 1L of media. You can split one bottle into multiple cultures, but keep in mind this will go through media faster. You can easily swap out bottles partway through an experiment to add additional media (or change the type of media) over time.

We like to inoculate each vial with 200-1000uL of culture, either in late exponential phase or overnight culture. Lower volumes can be used, but may lead to long lag time. Larger volumes should be avoided for convenience and to prevent contaminating the media input straw.

A user can pipette out up to 200uL with a long pipette tip at any point in the experiment (or up to 1mL by jamming one tip into another). Note that continuous cultures are much less dense than overnights, so consider this before downstream applications (DNA extraction, flow cytometry etc).

Data is collected every ~20s and OD and temperature can be observed remotely in real-time while the experiment runs using a server. Growth rate can be calculated in real-time and observed locally on the computer. Growth rate and other metrics (i.e. generations) can also be calculated after the fact using the .txt data files. Think about what information you’ll need to schedule things like timepoints or media switches.

Does anyone else have advice for first time users? What did you think about when converting your batch experiment to a continuous experiment?