Cytochrome expression: Aerobic growth in flasks

Authors: Heather Jensen, Cheryl Goldbeck,Caroline Ajo-Franklin
Last updated: 07/09/12
Goal: To express high levels of multi-heme cytochrome c in Esherichia coli

Materials needed:

  • Strains containing ccm promoters and the cytochrome c of interest
  • 14 mL round bottom falcon tubes, Falcon 352059
  • 2xYT, autoclaved: Note: do not use the “inert binder” type
  • appropriate antibiotics from group stocks
  • IPTG from group stocks
  • aminolevulinic acid (ALA), Sigma # A7793-1G

Plan Ahead: Sign up for incubator time and autoclave flasks.

Critical Steps:

There are a couple of factors that will strongly effect the expression level of c-cytochromes in E. coli. These include:

  • Strain of E. coli; ex: BL21(DE3) vs. C41(DE3)
  • Number of cytochromes expressed.
  • Promoter/RBS on expression plasmid and thus inducer concentration and time to induce
  • Promoter controlling cytochrome c maturation genes (ccmA-H)
  • Media; ex: LB vs. 2xYT
  • Temperature; ex: 30*C vs 37*C
  • Aeration due to shaking speed and surface area of media ex: 275rpm in 1.5mL deep well plate or 200rpm in a 250mL flask.


Day 1:

  1. Inoculate up 5 ml cultures in 2xYT plus antibiotic(s) from glycerol stock.

  2. Grow overnight 37 °C 275 rpm.

Day 2:

  1. Make up 1M stock solution of aminolevulinic acid in water.

    1. FW=167.59 g/mole

  2. Make up 2xYT Media, antibiotics, 1mM ALA minolevulinic Acid (1 mM).

  3. Typically, ~10am Use 1:100 dilution of overnight culture to inoculate 50 ml in 250 ml flask at 1/100 from O/N culture.

  4. Grow 30 °C 250 rpm to ~ O.D. 0.5-1.0. Record actual OD. Usually there by ~1-3pm.

  5. Induce by adding appropriate concentration 1 M IPTG.

  6. Grow overnight ~16 hrs. at 30 °C 250 rpm.

Day 3:

Collect FACS samples, Diffuse Reflection samples and/or take picture of pellets in plates


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