Team:Washington/alkanebiosynthesis

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Microbial Alkane Production Protocol

Contents


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


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

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


General Production Protocol

  • Grow 5mL of cells in Terrific Broth with Antibiotics overnight to saturation in 14mL culture tube
  • Measure OD (should be roughly 1.5)
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 0.7mL of Production Media with Antibiotic
  • Transfer to a 24mL 13x250
  • Seal the top with alluminum foil and grow at 37degC for 48 hours
  • 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