Endy:STN Chemostat Protocol

contact: Caitlin Conboy, Jennifer Braff

Day 0

Set up o/n culture in supM9 media in a small flask at 37 C.

Day 1

• Take OD600 of o/n culture. Dilute back in the morning into 25 mL/chamber supM9 (~1:100)
• When culture reaches ~OD 2.0 to add 20 mL to each chamber (usually late afternoon).
• Run at 37 C. The bubbler should be set to the white dot for 6 chambers (med/high) and the effluent needle should be set to around the 24 mL mark to maintain a volume of 20 mL.
• Add the media input (teal, 23-G1 needle) and turn on pump (currently 10 rpm for 20 mL/chamber). After about 1 hour, check that the culture volume is 20 mL and adjust effluent as necessary.
• The cultures should wash out to a stable OD600 by the morning (day 2). OD600 should be monitored throughout the day (~every 3 hrs) and samples drawn for steady-state measurement either in the latter part of day 2 or on day 3.

Notes

• Run with bleach solution, then H20 after use. Autoclave all components on fast (22 psi, 250 F, 30 min.).
• This water bath should be filled with dI. Top up frequently with dI kept at 37 C.
• Use 1_5_05 supplemented M9 minimal media with AMP (50 ug/mL) or Kan (20 ug/mL) as needed.
• Measure the flow rate by the effluent. 10 rpm gives about 0.25 mL/min, which is about right, but flow rate should be checked during each run.
• Mark 18 mL, 20 mL, and 22 mL on chemostat chambers to assist in monitoring volume.