Maitreya Dunham
Last revised December 2005
This comprehensive manual covers the entire process of running a chemostat, including media recipes, chemostat setup, inoculation, data acquisition and storage, daily monitoring, harvests for DNA and RNA, and data analysis. Although I wrote it with my own yeast cultures and ATR Sixfors setup in mind, many of the procedures are general. If you have edits or additions to the manual, please email me at maitreya@princeton.edu
Please feel free to point other people to these instructions. Also, I would appreciate the citation if you use any of this information in a publication or talk.
Visit genomics.princeton.edu/dunham for the most recent updates to the protocol and for my other protocols on microarrays.
Thanks to Matt Brauer, my long-time companion in the chemostat lab, for help developing these protocols and for proofing the manual. Also thanks to Frank Rosenzweig who taught me how to run my first glass-blown chemostats. Many of the protocols were influenced by his chemostat aesthetic.
Edits since last version: new phosphate limitation recipe, new DNA prep, new filter-sterilization method of media preparation
© Maitreya Dunham 2005
Contents................................................................................................ 2
Figures................................................................................................... 5
Planning the experiment.................................................................. 6
Signup....................................................................................................... 6
Strains....................................................................................................... 6
Limitations................................................................................................. 7
Dilution Rate.............................................................................................. 9
Chemostat media............................................................................... 11
Salts........................................................................................................ 11
10X salts for carbon,
leucine, or uracil limitation................................... 12
10X salts for phosphate
limitation........................................................ 12
10X salts for sulfur
limitation................................................................ 12
10X salts for nitrogen
limitation............................................................ 12
Metals...................................................................................................... 13
1000X metals........................................................................................ 13
Vitamins................................................................................................... 14
1000X Vitamins..................................................................................... 14
Tubing and Fittings.................................................................................. 15
Carboy..................................................................................................... 15
Autoclaving the salts............................................................................... 16
Additives.................................................................................................. 18
glucose (dextrose)
limitation additives................................................. 19
phosphate limitation
additives.............................................................. 19
sulfur limitation
additives...................................................................... 19
nitrogen limitation
additives................................................................. 20
leucine limitation
additives.................................................................... 20
uracil limitation
additives...................................................................... 20
Making a carboy of
media........................................................................ 21
Attaching the carboy................................................................................ 21
Plates...................................................................................................... 23
YPD....................................................................................................... 23
YNB....................................................................................................... 24
Supplements for YNB............................................................................ 25
canavanine plates................................................................................ 26
Dropout powder................................................................................... 26
5-fluorouracil plates.............................................................................. 27
alpha-aminoadipate
plates................................................................... 27
Kanamycin (aka G418 or
Geneticin) plates........................................... 27
Assembling the vessel............................................................................. 28
Autoclaving the
chemostats..................................................................... 31
Setting up a run................................................................................ 32
Polarizing the
dissolved oxygen probes.................................................. 32
Setting up the vessel............................................................................... 32
Connecting the air................................................................................... 33
Starting the media flow........................................................................... 33
Programming the
chemostat.................................................................... 35
Profiles.................................................................................................. 35
Calibrating the
Dissolved Oxygen Probe.................................................. 39
Calibrating the
temperature probe.......................................................... 40
Inoculating the
chemostats..................................................................... 40
Signup..................................................................................................... 42
Data collection......................................................................................... 43
Sample tracking....................................................................................... 45
Preparing to sample................................................................................ 45
Contamination issues.............................................................................. 46
Sampling.................................................................................................. 46
Klett...................................................................................................... 46
Glycerol stock........................................................................................ 47
Spectrophotometer............................................................................... 47
Sonicator.............................................................................................. 47
Coulter counter..................................................................................... 48
Plating for viable
counts....................................................................... 48
Plating for drug
resistance.................................................................... 49
Sampling for DNA.................................................................................. 49
Sorbitol Solution................................................................................... 49
Sampling for RNA.................................................................................. 49
Cleanup................................................................................................ 51
Counting colonies................................................................................. 51
Media Replacement.................................................................................. 51
Data Analysis.................................................................................... 54
Harvesting.......................................................................................... 55
Setup for harvesting................................................................................ 55
Harvesting............................................................................................... 56
Sample Processing........................................................................... 59
Filtrates................................................................................................... 59
Culture revival......................................................................................... 59
DNA prep................................................................................................. 60
RNA prep.................................................................................................. 60
Lysis buffer........................................................................................... 62
RNA prep for 5 ml daily
samples............................................................ 63
RNA prep for 100 ml
harvest................................................................. 64
Problems............................................................................................. 65
Chemostat References.................................................................... 67
Supplies and Suppliers.................................................................. 68
ATR....................................................................................................... 68
Fisher.................................................................................................... 68
GE Osmonics......................................................................................... 69
Ace Glass.............................................................................................. 70
Masterflex/Cole Parmer........................................................................ 70
R-Biopharm AG...................................................................................... 71
McMaster-Carr...................................................................................... 71
Figure 1. Testing the limitation in batch.................................................... 8
Figure 2. Testing the limitation in the
chemostat...................................... 9
Figure 3. Tubing and fitting menagerie................................................... 15
Figure 4. Foil origami............................................................................... 17
Figure 5. Assembled media carboy......................................................... 18
Figure 6. Assembled chemostat vessel................................................... 30
Figure 7. Rubber band pump trick........................................................... 35
Figure 8. Status screen........................................................................... 36
Figure 9. Fermenter menu...................................................................... 36
Figure 10. Edit profile 0........................................................................... 37
Figure 11. Edit profile 1........................................................................... 38
Figure 12. Inoculated chemostat............................................................ 41
Figure 13. Header index card.................................................................. 44
Figure 14. Sampling index card............................................................... 44
Figure 15. Small filtering apparatus........................................................ 50
Figure 16. Media conservation trick........................................................ 52
Figure 17. Large filter apparatus............................................................ 56
The first thing to do is design your experiment. You need to choose a strain, a limitation, a limiting nutrient concentration, and a dilution rate. You also need to arrange to use the chemostats.
Once you've got an experiment planned, sign up on the board by the chemostat room. That way, other people can plan their own chemostat use.
The strains commonly used in the lab are FY, which is an S288C derivative that's been made GAL2+, and CEN.PK, a favorite of the European chemostat community. Using a prototroph is vastly preferred to using an auxotroph. With auxotrophs, you can never really be sure what the cells are using as a source of limiting nutrient. It just complicates matters and makes you less sure of any results. We have prototrophs of FY and CEN.PK, as diploids and as haploids of both mating types, in the strain collection:
|
Background |
Mating type |
AKA |
|
|
FY |
a/alpha |
FY4xFY5 |
MD collection |
|
FY |
a |
FY4 |
11069 |
|
FY |
alpha |
FY5 |
11070 |
|
CEN.PK |
a/alpha |
|
9500 |
|
CEN.PK |
a |
|
11092 |
|
CEN.PK |
alpha |
|
11093 |
The FY haploid strains are from Fred Winston. The CEN.PK diploid, DBY9500, is direct from Peter Kotter. The FY diploid and the CEN.PK haploids were derived in my lab by mating and dissection, respectively.
Both strain backgrounds grow well in glucose, phosphate, and sulfur limitation. I have had very odd results with FY in nitrogen limitation, although CEN.PK seems to behave. All S288C derivatives have a Ty element in HAP1 that decreases its activity. We now also have a HAP1+ derivative of FY from Fred Winston's lab. CEN.PK strains have a mutation in CYR1. Also, LEU2 may not be in the usual location.
You can, of course, use other strains. The biggest unknown danger of a new strain is its flocculation capacity. Because they can stick to the vessel and sink to the bottom to avoid being diluted out, clumpers are selected for in the chemostat. In addition to complicating cell count data, they also make it very difficult to understand what's going on in terms of selection pressure, clonal selection, etc., so the chemostat run is effectively over once they appear. For CEN.PK and FY, I've gotten clumpers occasionally ~400 generations into the evolutions. Many lab strains carry knock outs of several FLO genes, making the transition to flocculation difficult (although some of the knock out mutations, like the one in FLO8 in S288C, are point mutations that may revert). Other strains, such as SK1, flocculate even in rich media, making them next to useless in the chemostat. When using a new strain, be particularly vigilant about frequently checking the culture with the microscope before and after sonication. If sonication effectively breaks up the clumps, I don't think it's a serious enough problem to halt the chemostat, although I would make a note of the phenotype in my log. For short-term cultures, this is not nearly as much of a problem, so you have a wider variety of strains available.
If you do have to use an auxotroph, be very careful with the supplements you add. For example, you can't use adenine sulfate with sulfur limitations. You want to make sure the culture does not become limited for the additive, but you don't want to add so much excess that the culture eats the additive instead of the nominal limiting nutrient. See the Limitations section for how to check limiting nutrients. You can also use an auxotroph on purpose and limit with the additive it requires. Matt Brauer and Alok Saldahna have successfully done this with leu2 strains, and Alok and I have also done ura3 strains. I've included these media formulations in the Media Recipes section.
The limiting nutrient depends on what your experiment is. Keep in mind that glucose limited cultures seem to be most sensitive to changes in the dilution rate. Lower dilution rates provoke more respiration while higher dilution rates favor fermentation. Nitrogen limitation does not work well with FY.
If you are not using one of the standard recipe/strain combinations that I list in the Chemostat Media section, you should do a preliminary experiment to figure out the limiting concentration to use. These experiments are conveniently done in batch. Inoculate an overnight culture. Spin it down and resuspend at a 100X dilution in chemostat media without any limiting nutrient. Aliquot equal volumes into a series of appropriate volume shake flasks that contain different quantities of the limiting nutrient. Be careful that the volumes of limiting nutrient solution are the same in all the flasks so you don't get different dilution factors. You may want to make your media 1.1X and bring them to 1X with the limiting nutrient solution. Let these flasks shake at 30C for a couple of days or until the density stabilizes. You want them to be completely in stationary phase. Measure the densities. If you graph the concentration of limiting nutrient vs. the final densities of the cultures, you should get