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
References:

• Jensen, et. al. (2010) Engineering of a synthetic electron conduit in living cells. PNAS; SI information: http://www.pnas.org/content/suppl/2010/10/12/1009645107.DCSupplemental/pnas.1009645107_SI.pdf#STXT
• “Expression of recombinant cytochromes c in E. coli” Chapter by Yuri Londer in Methods Mol. Bio: http://www.ncbi.nlm.nih.gov/pubmed/21125384
• “Tuning promoter strengths for improved synthesis and function of electron conduits in Escherichia coli.“ C. P. Goldbeck, H. M. Jensen, M. A. TerAvest, N. Beedle, Y. Appling, M. Hepler, G. Cambray, V. Mutalik, L. T. Angenent, C. M. Ajo-Franklin. (2013) ACS Synth. Biol., 2(3), 150-9 (2013).

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.

Procedure

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