The overall goals of the following presentation are to describe the principle of the chemos stat, demonstrate their operation, and to provide examples of applications that use chemos stats. This is achieved by first assembling the hemostat vessel inoculating, and then continuously adding new media while removing old media and cells at a constant rate. Mathematical modeling describes the change in cell density and the change in concentration of a growth limiting nutrient within each hemostat.
The use of these equations allows for the prediction of a single non-zero stable steady state at which cell and nutrient concentrations are constant, and the specific growth rate of cells is equal to the rate at which the cells are removed. By combining the precise control of cell growth and the environment within the chemos stats with other technologies results that enable systems level investigations into cell growth regulation, genetic changes and adaptive evolution are obtainable. The main advantage of this method of cell culturing over other techniques is that the growth rate is controlled and the cell's external environment remains constant.
This method can help answer key questions in the fields of cell biology and evolution, such as what is the molecular basis of cell growth regulation and adaptive evolution in defined environments. Visual demonstration of this method is critical as setting up and maintaining hemostat cultures is difficult to the large number of steps and components involved. Begin this procedure by first turning on the six four system using the main switch.
Next, thoroughly rinse the hemostat vessel, stir assembly, and attached tubing with deionized water. Check all the tubing and O-rings and replace any worn out looking pieces. Once the stir assembly and drive shaft have been assembled, fill the vessel with 300 milliliters of DI water.
Check the electrolyte level of the dissolved oxygen probe, making sure that the bottom casing is filled halfway with electrolyte solution. Next, calibrate the pH probe. Once calibrated, detach the probe from the fermentor.
Rinse with DI water and insert it into the port on the hemostat vessel. Screw until finger tight With the probes connected, place the hemostat vessel in the rack, making sure to note on the vessel which fermentor the pH probe was calibrated with. Finally, prepare the probes and tubing for autoclaving by placing the cap on the pH probe and cover the dissolved oxygen probe and all free tubing ends with foil autoclave.
An empty car that is capped with a rubber plug containing an air inlet and media outlet. Ensure that the end of the media tubing is covered in foil. Attach a 500 milliliter filter cup to a 100 milliliter media bottle.
Remove the cotton filter from the filter with forceps, sterilized and ethanol, and immediately attach to the filtration port on the media car. Attach the media car to a vacuum source and filter. Sterilize 10 liters of freshly prepared media by adding it to the filter cup.
When filtration of media is complete, close off the filtration port with a metal clamp. After the hemostat vessels have been autoclave and allowed to cool down, place the vessel in its corresponding heat jacket. Connect the temperature, probe pH probe and dissolved oxygen probe.
Let the vessel sit with power on for at least six hours to allow the dissolved oxygen probes to polarize eyes. Then connect the media carbo to the hemostat vessel. Use ethanol in order to keep the tube ends as sterile as possible.
Then thread the pump tubing through the pump and open the clamp. Next, place the end of each effluent tube into a separate collecting receptacle. Connect the air supply via an autoclave filter and turn on airflow.
The water in each muscle should flow out of the effluent tubes, which indicates that all seals are properly formed. Adjust the height of the effluent tube to the desired working volume of 300 milliliters. Manually press the pump until the media starts to flow into the hemostat vessel.
Then release the tubing from the pump. When the media reaches the effluent tube, reattach tubing to pump and clamp with the media primed. Start running the basic program within impeller set to 400 RPM and the temperature set at 30 degrees Celsius.
Next, calibrate the dissolved oxygen probes by turning off the air supply and switching to nitrogen gas. Wait at least one hour and record the measured value as low read. Switch back to an air supply containing an ambient oxygen concentration and wait at least one additional hour.
Record this measurement as the high read value. At this point, initiate data recording using iris software prior to inoculation. Sterilize the top of the culture vessel with 70%ethanol.
Then open the vessel port using a screwdriver and pipette, one milliliter of an overnight culture into the vessel, replacing the screw when finished. Once the cultures reach their early stationary phase, initiate the pumps and establish a desired flow rate. By specifying the number of seconds the pump is on and off, the flow rate and culture volume.
Determine the dilution rate. Using the six fours interface, define a program that specifies the pump timing temperature and impeller speed. Then start the program after at least two hours.
Record the time and use a graduated cylinder to measure how much media has been removed from the vessel. The amount removed will equal the volume that has been added to the hemostat vessel and can be used to calculate the dilution rate, sap the outflow, and use it to measure cell density. Steady state growth is achieved when cell density does not change over time.
Once a steady state has been achieved, acquire small samples by taking them from the effluent tube over the course of a few minutes by letting it passively flow into a collection tube. Gene expression and metabolite analyses require larger samples. These can be quickly acquired by releasing the screw, holding the metal end of the effluent tube, and slowly lowering the tube.
When a large volume is removed, the flow rate must be reduced to maintain the same dilution rate. First, establish steady state cultures of two yeast strains with identical dilution rates and volumes passively Sample one milliliter from each vessel to be used as controls for subsequent flow cytometry analysis. Spin down the cells for one minute at 6, 000 RPM, resuspend them in one milliliter of PBS and store the tube at four degrees Celsius.
Next, place the effluent tube from one vessel at a time into an autoclave graduated cylinder. Release the screw holding the effluent tube and gently push down to expel 150 milliliters of cells from the chemos stat, returning the metal end of the effluent tube to its original position. When finished, after sterilizing the area, open the vessel port on the top of the chemos stat vessel.
Place a sterile funnel in the opening and add 150 milliliters from the other culture. Then close the port and repeat with the second vessel. Each vessel now contains a 50 50 mixture of the two strains, possibly acquire samples every two to six hours for two to three days.
Store samples in PBS and analyze strain proportions in each sample using flow cytometry. After establishing a steady state hemostat culture using a strain of known genotype with a defined nutrient limitation, maintain the unperturbed culture for several hundred generations, replenishing the media reservoir with fresh media as necessary. Maintain a fossil record of the adapting population by passively sampling the outflow every two to three days.
Store the sample in 15%Glycerol at negative 80 degrees Celsius is after completion of the selection, played a sample of cells on solid agar plates. Once cells have grown into colonies, pick an unbiased sample of colonies using toothpicks and inoculate clones into individual wells of a 96 well plate containing 100 microliters of media. Allow clonal samples to grow overnight.
Then add 100 microliters of 30%glycerol to each well and store at negative 80 degrees Celsius until used for genomic analysis. A major advantage of chemos stats is the ability to control the growth rate of cells. The percentage of yeast cells, which are budding shown here on the Y axis is a good representation of their growth rate.
As seen here. This percent increases in a near linear manner with increased dilution rate. Faster dilution rates result in faster growth rates, which may lead to systematic increases in gene expression, represented here by an increase in UTR two mRNA abundance.
However, systematic decreases in gene expression may also occur with increased growth rate, as in the case of ASM one. The relative fitness of different genotypes can be determined by conducting competition assays in hemostats using fluorescently labeled cells and flow cytometry analysis shown here is a representative result in which a mutant was competed against a fluorescently tagged wild type strain. In this example, the mutant strain has a 40%growth rate advantage per generation.
Whole genome sequence analysis can be used to identify acquired mutations in cells with increased fitness, following long-term selection in chemos stats. For example, analysis of independent adaptive evolution experiments in nitrogen limiting conditions, identified different copy number variant alleles at the gap, one locus, which encodes the general amino acid permease. Following this procedure, other methods such as mass spectrometry, flow cytometry, and next generation sequencing can be used in order to answer similar questions regarding systems level regulation of cell growth and adaptive evolution.
After the development of this technique has paved the way for researchers in fields from microbiology to cell biology to study how cell physiology and molecular processes are affected by variation in cell growth rate. After watching this video, you should have a good idea of how to set up and maintain chemo set cultures, as well as the different ways in which samples are required for different applications.
