Team:Calgary/BetaOxidation

☰

• Home
• Project
  • Problem/Description
  • Synthesis
    • Synthesis Overview
    • Beta Oxidation
    • Glycolysis
  • Secretion
  • Process
    • Process Overview
    • VFA Fermentation
    • Solid-Liquid Separation
    • PHB Fermentation
    • Extraction
    • Products
  • Applied Design
  • Experiments
  • Results
  • Composite Parts
  • Modelling
• Notebook

• 

• Team
  • Team Members
  • Attributions
  • Sponsors
• Human
  Practices
  • HP Summary
  • HP Silver
  • HP Gold/Integrated HP
  • Collaborations
  • Engagement
  • Safety
• Judging
  • Medal Requirements
  • Interlab

Beta Oxidation

Insert beta-oxidation pathway only image here

Aim

...bla bla bla

Volatile fatty acids & beta-oxidation pathway

…

Gene construct

Insert gene construct image here

…

Chassis and vector

…

Experiment

Glucose (Positive control) 10 ml O/Ns in LB+Kan 7 ml 20% Glucose {..!!cite why this conc chosen..} 100 uL MgSO4 5 uL CaCl2 10 ml M9 salts 5 uL 1M IPTG 20 ml dH2O	pET29B in BL21 (Negative control) 10 ml O/Ns in LB+Kan "Syn poo" fermented supernatant {..!!hyperlink to eng page for syn poo protocol..} 100 uL MgSO4 5 uL CaCl2 10 ml M9 salts 5 uL 1M IPTG
Fermented "syn poo" supernatant 10 ml O/Ns in LB+Kan 10 ml "Syn poo" fermented supernatant {..!!hyperlink to eng page for syn poo protocol..} 100 uL MgSO4 5 uL CaCl2 10 ml M9 salts 5 uL 1M IPTG 23 ml dH2O	Pure VFAs 10 ml O/Ns in LB+Kan VFAs 410 uL propionic acid 118 uL acetic acid 55 uL butyric acid 100 uL MgSO4 5 uL CaCl2 10 ml M9 salts 5 uL 1M IPTG 30 ml dH2O

The O/Ns were grown in the respective media for ~24 hours. The flasks containing different media was inoculated with the O/Ns after adjusting the OD₆₀₀. The composition of each of the replicate in the flasks is shown below:

pET29B in BL21 (Negative control)

• 10 ml O/Ns in LB+Kan
• "Syn poo" fermented supernatant {..!!hyperlink to eng page for syn poo protocol..}
• 100 uL MgSO₄
• 5 uL CaCl₂
• 10 ml M9 salts
• 5 uL 1M IPTG

{..........what else was added was supernatant added....}

Glucose (Positive control)

• 10 ml O/Ns in LB+Kan
• 7 ml 20% Glucose {..!!cite why this conc chosen..}
• 100 uL MgSO₄
• 5 uL CaCl₂
• 10 ml M9 salts
• 5 uL 1M IPTG
• 20 ml dH₂O

Fermented "syn poo" supernatant

• 10 ml O/Ns in LB+Kan
• 10 ml "Syn poo" fermented supernatant {..!!hyperlink to eng page for syn poo protocol..}
• 100 uL MgSO₄
• 5 uL CaCl₂
• 10 ml M9 salts
• 5 uL 1M IPTG
• 23 ml dH₂O

Pure VFAs

• 10 ml O/Ns in LB+Kan
• VFAs
  • 410 uL propionic acid
  • 118 uL acetic acid
  • 55 uL butyric acid
• 100 uL MgSO₄
• 5 uL CaCl₂
• 10 ml M9 salts
• 5 uL 1M IPTG
• 30 ml dH₂O

The OD₆₀₀ readings of .....???? were taken and recorded:

Condition	OD600 of replicate 1	OD600 of replicate 2	OD600 of replicate 3
pET29b in BL21 (Negative control)	0.571	0.531	0.487
Glucose (Positive control)	0.190	0.195	0.139
Pure VFAs	0.140	0.134	0.146
Fermented "syn poo" supernatant	0.135	0.107	0.144

After spinning down the culture in flasks, the cells were resuspended in 1x PBS. The OD₆₀₀ readings were taken:

Condition	OD600 of replicate 1	OD600 of replicate 2	OD600 of replicate 3
pET29b in BL21 (Negative control)	2.659	2.001	2.899
Glucose (Positive control)	1.934	1.887	1.919
Pure VFAs	0.510	0.571	0.532
Fermented "syn poo" supernatant	2.533	2.559	2.349