McClean:Chemostat Metabolic Cycle
This is a guide on how to induce the Metabolic Cccle in FY diploid Yeast Strains (First used on 8/5/11-8/14/11) and subsequently sample the culture for various things. To see how to set up a chemostat see McClean Lab Chemostat Guide.
Inducing the Cell Metabolic
Relevant Chemostat Profiles
Profile 0: RPM - 400 Affluent Tube - Off pH - Off Temp - 30 °C DO - minimal
Profile 1: RPM - 400 Affluent Tube - On pH - Off Temp - 30 °C DO - minimal
For Dilution Rate .08 hr-1 : 2/20 For Dilution Rate .12 hr-1 : 3/20
- Fill the vessel with 300 mL of Media. If the Media doesn't automatically fill in the chemostat when the air pressure is turned on and the affluent tube is connected, connect a large tube to the sterile filter of the media carboy and blow some air into it to increase the pressure. Make sure the affluent tube is UNCLIPPED so it can fill.
- Set the profile to 'Profile 0' and then start the program.
- Using 70% ethanol to sterilize, open one of the valves on the top (use a screw driver if ineccessary). Quickly add 3 mL of Overnight Culture in YPD of the strain of interest.
- Quickly tighten the screw and leave the culture as is.
- After ~48 hours (24 hours to reach saturation and 24 hours for starvation) turn off the chemostat program. Change to Profile 1, and restart the chemostat.
- Wait 10-16 hours and look for a characteristic cycling profile (image coming soon). The period and amplitude should be fairly constant.
- Make sure that the pattern is maintained for multiple cycles before confirming the cycle. Sometimes cultures can begin to oscillate, but then lose the amplitude and go back to baseline. In the event of this, or any such perturbation that causes cycling to be lost, change the chemostat to profile 0 and restarve for 48 hours.
- The cycling, once started, should last as long as none of the normal settings are drastically changed.
Cells can be sampled directly from the chemostat for a variety of reasons such as checking density, extracting mRNA, checking budding index, or imaging cells using FISH or IF or Fluorescent Microscopy. For a more in depth guide see the Botstein Lab Chemostat Guide . Here we will explain the protocl for sampling cells for fixing cells in formaldehyde.
- Note: It is important that you sample an appropriate volume. You don't want to oversample at any give time point or over a certain period of time in such a way that the culture cannot recover. For a 300 mL culture at a dilution rate of .08 to .12 hr-1<\sup> we sampled 900-1000 uL every 20 minutes: this is about 1/10 of the volume that is replaced in this time period.
- Fill 1.5 mL eppendorf tube with 100 uL of 32% paraformaldehyde (IN THE HOOD). It's easier to aliquot all these tubes at once. Make sure to label the tubes well!.
- When it comes time to sample from a chemostat put on gloves and wipe down one of the sealed valves in ethanol. Make sure to wear gloves too, getting the culture contaminated would bad.
- Quickly take of the top of the valve and set it on top of the vessel.
- Using a 2mL serological pipette and a hand controlled (not a motorized) pipetteor, remove 900-1000 uL of culture directly from the open vessel.
- Replace and tighten the cap.
- Add culture up until the 1 mL line on the eppendorf tube. Close and invert several times. The remaining culture can be used to image in a 96 well plate or to count bud index etc. Alternatively many of these measurements can be done later on fixed cells.
- Keep the tubes at room temp for 1 hour and then place in a 4°C box.
- The samples can be imaged or used as is, or can be spheronblasted for IF or FISH.