From 2011.igem.org

Microbial Alkane Production Protocol

Current Protocol for 100mL of Media

Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Microbial Biosynthesis of Alkanes Science Report]

Long Term Stocks to Prepare

Store at room temperature unless otherwise noted

• 1L of 1M Bis‐Tris (pH 7.25)
• 10mL of 1mg/mL Thiamine (store at -20 in 1mL aliquots)
• 10mL of 10% Triton X-100
• 1M MgSO4
• 0.1M FeCl3-6H2O
• Trace Metals
  • 1M CaCl2
  • 1M MnCl2-4H2O
  • 1M ZnSO4-7H2O
  • 0.2M CoCl2-6H2O
  • 0.1M CuCl2-H2O
  • 0.2M NiCl2-6H2O
  • 0.1M Na2MoO4-2H2O
  • 0.1M Na2SeO3-5H20
  • 0.1M H3BO3

100mL M9 minGlucose Media Prep

ADD IN ORDER, make sure you have a sterilized Erlenmeyer flask for the initial mixing and a sterilized bottle to sterile filter into

Constantly mix using a stir bar

• 75mL ddiH2O
• 3g glucose (Final 3%, 100% = 1g/mL)
• 0.6g Na2HPO4
• 0.3g KH2PO4
• 0.05g NaCl
• 0.2g NH4Cl
• 20mL of 1M Bis-Tris (pH 7.25)
• 1mL of 10% Triton
• 100uL of 1mg/mL thiamine
• 100uL of FeCl3-6H2O
• 100uL of MgSO4
• 5uL of Each Trace Metal (do not mix together before, they crash out)

Once media is prepared sterile filter into a pre-sterilized glass bottle

Aliquot 0.7mL into culture tubes and add appropriate antibiotic (1microL of Kan or Chlor)

Pick colonies and inoculate cultures, but DO NOT eject the tip into the tube

Grow cultures for 40+ hrs at 37degC (if inducible add 1mM IPTG at and OD600 of ~1.0)

Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an eppindorf tube, and spin at max speed for 1minute.

Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert

Run sample on GC-MS and identify Alkanes