Dunham Lab Chemostat Manual

 

Maitreya Dunham and Emily Mitchell

Last revised December 2010

 

 

 

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 it was written with our blown-glass chemostats in mind, many of the procedures are general, and the principles could be applied to other systems.  If you have edits, additions, or suggestions for the manual, please email me at maitreya@uw.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 http://dunham.gs.washington.edu for the most recent updates to the manual and for my other protocols on microarrays and yeast genetics.  Also available is the old version of the chemostat manual, which covers use of ATR Sixfors Fermenters.  That portion of the manual has forked and is now maintained by the Botstein lab.

 

Thanks to Matt Brauer, my long-time companion in the chemostat lab, for help developing these protocols.  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.  Finally, many members of the Botstein and Dunham labs contributed improvements.

 

Edits since last version: My new set up at the University of Washington employs blown glass chemostats, and so this manual has been updated to focus on their use and care.

 

 

copyright Maitreya Dunham 2010

Table of Contents

 

Planning the experiment...................................................................... 5

Signup................................................................................................... 5

Strains.................................................................................................. 6

Table 1. Yeast strains for the chemostat............................................................... 6

Limitations............................................................................................. 7

Figure 1. Testing the limitation in batch................................................................. 8

Figure 2. Testing the limitation in the chemostat.................................................. 9

Dilution Rate.......................................................................................... 9

Chemostat media................................................................................. 11

Salts................................................................................................... 12

10X salts for phosphate limitation (1 L)............................................... 13

10X salts for sulfur limitation (1 L)...................................................... 13

10X salts for nitrogen limitation (1 L).................................................. 13

Additives.............................................................................................. 14

Metals............................................................................................. 15

Vitamins.......................................................................................... 16

Setting up a run.................................................................................. 17

Mixing and Filtering the media.................................................................... 17

Prepping a new Carboy...................................................................... 18

Figure 3. Setting up the carboy............................................................................. 18

Foil Origami..................................................................................... 19

Figure 4. Foil Origami............................................................................................ 19

Preparing a Carboy for Media............................................................. 20

Fetching Water................................................................................. 20

Mixing the Media.............................................................................. 21

Filtering the Media............................................................................ 21

Ready, set, filter:........................................................................................................ 22

Figure 5. Getting ready to filter, The Carboy........................................................ 22

Figure 6. Setting up the filter................................................................................. 23

Figure 7. Final assembly of filter set up................................................................ 24

Figure 8. Turn Vacuum on LOW............................................................................ 25

Figure 9. During filtration....................................................................................... 26

About the Chemostats......................................................................... 27

Figure 10. The three systems of a chemostat..................................................... 27

Figure 11. Anatomy of a Chemostat..................................................................... 28

Setting up the Chemostats.................................................................. 28

Prepping a new chemostat.......................................................................... 29

Table 2. Working volumes of Unaltered Chemostats........................................... 30

Figure 12. Downward angle of effluent track......................................................... 31

Clearing the way for aeration.................................................................... 31

Cleaning the Bubblers....................................................................... 32

Baking the frit.................................................................................. 32

Setting up a Chemostat for a Run................................................................ 33

Figure 13. Set up new pump tubing....................................................................... 34

Figure 14. Two chemostats running off of one carboy......................................... 35

Filling the chemostats........................................................................ 36

Inoculation....................................................................................... 37

Starting the pumps........................................................................... 37

Setting the Pump Rate............................................................................................... 37

Table 3. Example Pump rates for Chemostats..................................................... 38

Loading the Pump Heads........................................................................................... 38

Starting the Pump...................................................................................................... 39

Figure 15.  Dummy lines........................................................................................ 40

Sampling the Chemostats.................................................................... 41

Figure 16. Taking a sample during a run.............................................................. 41

Preparing to sample................................................................................. 42

Figure 17.  Example of Minimal sampling......................................................... 43

Figure 18. Experiment Layout Sheet.................................................................... 43

Figure 19. Header index card................................................................................ 44

Figure 20. Sampling index card............................................................................. 44

Sample tracking................................................................................ 45

Contamination issues........................................................................ 45

Figure 21. Checking for media line occupancy.................................................... 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

Figure 22. Small filtering apparatus...................................................................... 50

Cleanup after sampling..................................................................... 51

Counting colonies............................................................................. 51

Example of daily sampling................................................................. 51

Sample Analysis During a Run..................................................................... 52

D = effluent volume/(time * chemostat volume)...................................................... 52

Media Replacement.................................................................................. 53

Harvest............................................................................................... 54

What is 'Steady State'.............................................................................. 54

How long should Evolutions go?................................................................. 55

Setup for harvesting............................................................................... 55

Figure 23. Large Vac kit........................................................................................ 56

Harvesting............................................................................................ 56

Figure 24. 'Harvest Cart' complete with the large filter apparatus..................... 57

Cleanup............................................................................................... 58

Taking down chemostats:.................................................................. 59

Taking down all the chemostats:.............................................................................. 61

Sample Processing.................................................................................. 61

Chemostat Troubleshooting.............................................................. 63

Appendix A:  Sample processing........................................................ 65

Culture revival...................................................................................... 65

Coulter Counter Instructions................................................................... 66

Troubleshooting the Coulter Counter................................................... 68

Testing Filtrates.................................................................................... 69

 

DNA prep.............................................................................................. 70

Lysis buffer for DNA........................................................................... 71

RNA prep.............................................................................................. 72

Lysis buffer for RNA........................................................................... 72

RNA prep for 5 ml daily samples............................................................................... 73

RNA prep for 50 ml harvest....................................................................................... 74

Appendix B:  If we had it to do over again...................................... 75

The Disaster Index-Where you don't want to be............................................... 75

Cork Sucking.................................................................................... 75

Figure 25. Hacking at the Cork............................................................................. 75

Pump problems................................................................................ 75

Media line contamination................................................................... 76

Figure 26. Major media line occupation................................................................ 76

Breakage......................................................................................... 76

Changes to the chemostat design................................................................. 77

Consistent chemostat volumes........................................................... 77

Table 4.  Distances between frit and overflow...................................................... 77

Media line dropper............................................................................ 77

Appendix C: Parts and Suppliers....................................................... 78

Tubing and Fittings................................................................................. 78

Figure 27. Tubing and fittings............................................................................... 78

Supplies and Suppliers.............................................................................. 79

Chemostat References........................................................................ 85

 

 


Planning the experiment

 

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.  Figure out how much media you'll need, and arrange to use the chemostats.  Don't try to sign up now, and figure it out later, because you'll end up regretting it.  Take the time to read through the manual, and understand the full experiment before you sign up.

 

When you've read through the manual, arrange to speak with someone in the lab who is practiced with the chemostats, so they can help you.  For your first chemostat experiment, plan to run no more than 4 chemostats, and coordinate with someone for setting up, starting the run, and sampling.  Then you'll be pretty much on your own until the end of the experiment, when you'll want someone to go over the clean up/ recovery stage.

 

Signup

 

Once you've got an experiment planned, sign up on the Chemostat sign-up calendar.  That way, other people can plan their own chemostat use.  List your name, which chemostats you'll want to use, and for how long.  Include time for preparation and clean up in your timeline. 

 


Strains

 

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

DBY number

FY

a/alpha

FY4xFY5

YMD 132

FY

a

FY4

DBY 11069

FY

alpha

FY5

DBY 11070

CEN.PK

a/alpha

 

DBY 9500

CEN.PK

a

 

DBY 11092

CEN.PK

alpha

 

DBY 11093

Table 1. Yeast strains for the chemostat.

 

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. Oddly, CEN.PK seems to behave better than FY in nitrogen limitation.  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, and we have.  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, frequently flocculate, 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, it's probably not a serious enough problem to halt the chemostat, although you should make a note of the phenotype in your 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 Saldanha have successfully done this with leu2 strains, and Alok and I have also done ura3 strains. These media formulations are included in the Media Recipes section.

 

 

Limitations

 

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 in my hands, though others have had more success.

 

If you are not using one of the standard recipe/strain combinations listed in the Chemostat Media section, you should do a preliminary batch culture experiment to figure out the limiting concentration to use.  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 a plot with a linear range, a nonlinear range, and a plateau.  You want to stay in the linear range.  Here's representative data from one of the experiments for phosphate limitation:

Figure 1. Testing the limitation in batch.

Note that the Y-intercept is not zero.  That's probably from nutrient the yeast stored from the overnight culture, which was in rich media in this experiment.  Take that into account when you decide on a chemostat media concentration that will yield your desired density. We aim to run our evolutions starting from a population size of 3-5x107 cells/ml.  To measure the final density more exactly, once you have a good idea of what concentration is limiting, grow the first overnight in limiting chemostat media.  The cells will use up all the limiting nutrient and the zero will really be zero.

 

Although the batch results generally match the chemostat quite well, make sure to test the concentration in the chemostat to double check that it's really limiting.  As a cautionary tale, we lost over a year of work from failing to properly do this control: a typo in the phosphate limitation media recipe resulted in a low potassium concentration, an error that had no effect on the batch culture results yet profoundly affected the chemostat cultures.  To convince yourself that your chemostats are limited by what you think they are, run 2 chemostats to steady state.  Once they've hit steady state, in one chemostat, increase the nominal limiting nutrient in the feed media by 50% and watch for an increase in density.  In the other, increase the sugar, vitamins and metals by 50% and see if the density changes.  If you are truly limited only for the limiting nutrient, you will see ~50% increase in density in the first one but no change in density in the second one.  Density is actually not the greatest indicator since it's really yield you're interested in, but it usually does track pretty well.  Klett seems to work the best as a surrogate for yield.

 

In this example from David Hess, six cultures were grown to steady state with 20 mg/L potassium phosphate.  At the indicated point, the feed media was switched to 30 mg/L potassium phosphate.  F13, F15, and F17 are all limited by phosphate.  F14 and F16 are not.  F18 washed out.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 Figure 2. Testing the limitation in the chemostat.

 

Also note that not all strains behave exactly the same.  In some circumstances, it may be easier to run two separate chemostats, each with a different (supposedly limiting) concentration of the nutrient.  Then compare the steady state densities.  This experimental design is particularly useful when the strains take a long time to hit steady state and so evolution is a concern.

 

Dilution Rate

 

We most typically use a dilution rate of 0.17 (+/-) 0.01 chemostat volumes per hour.  The Paquin and Adams experiments were all at 0.2 chemostat volumes per hour.  You will know if you set the dilution rate too high (i.e., above the maximal growth rate) because your culture will wash out.  In glucose limitation, there is a critical dilution rate where the culture switches from respirofermentative growth to primarily fermentative growth.  Growth-rate dependent changes have been studied in great detail by the Botstein, Oliver, and Regenberg labs.

 

The dilution rate is a simple relation of the effluent volume, length of time (in hours) effluent collected, and chemostat volume:

 

D = effluent volume/(time * chemostat volume)

 

The dilution rate is in units of hr-1.  It is also sometimes called omega.


Chemostat media

 

Chemostat media has 4 components that need to be made separately:  salts, metals, vitamins, and carbon/sugar.

 

For each batch of media, you will prepare a carboy, thaw the 1000X vitamins, make 10X salts, and make 10X carbon source.  You'll combine these with the pre-made metals and vitamins, and top off to 10L with glass distilled water.  It all gets mixed together in a non-sterile "mixing" carboy before it gets filtered into a sterile carboy.

 

These media recipes come from Julian Adams via Frank Rosenzweig with further modification by me.  The glucose limitation recipe is exactly per Adams.  I modified the glucose limitation recipe for phosphate, sulfur, and nitrogen limitation.  In general, I tried to keep all ions at the same molarity where possible.  The Adams version of the phosphate limitation recipe uses the salts at 0.25X to limit the effects of phosphate contamination from the other salts, but I always use 1X salts for everything.  You can only get away with this if you use really pure chemicals.