Team:Washington/alkanebiosynthesis

From 2011.igem.org

(Difference between revisions)
(Created page with "=Microbial Alkane Production Protocol= ==Current Protocol for 100mL of Media== Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Micro...")
 
(10 intermediate revisions not shown)
Line 1: Line 1:
 +
{{Template:Team:Washington/Templates/Top}}
 +
 +
=Microbial Alkane Production Protocol=
=Microbial Alkane Production Protocol=
 +
 +
__TOC__
 +
 +
==Current Protocol for 100mL of Media==
==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]
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'''===
 
 +
==='''Long Term Stocks to Prepare'''===
Store at room temperature unless otherwise noted
Store at room temperature unless otherwise noted
*1L of 1M Bis‐Tris (pH 7.25)
*1L of 1M Bis‐Tris (pH 7.25)
Line 11: Line 18:
*1M  MgSO4
*1M  MgSO4
*0.1M FeCl3-6H2O
*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
 
Line 38: Line 35:
*100uL of FeCl3-6H2O
*100uL of FeCl3-6H2O
*100uL of MgSO4
*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
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
+
==='''General Production Protocol'''===
-
Run sample on GC-MS and identify Alkanes
+
*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 Eppendorf tube
 +
*Spin down cells at 4000rpm for 10 minutes
 +
*Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL Eppendorf 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 Eppendorf 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

Latest revision as of 00:20, 29 September 2011


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 Eppendorf tube
  • Spin down cells at 4000rpm for 10 minutes
  • Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL Eppendorf 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 Eppendorf 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
Retrieved from "http://2011.igem.org/Team:Washington/alkanebiosynthesis"