Beta Oxidation

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Aim

...bla bla bla

Volatile fatty acids & beta-oxidation pathway

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Gene construct

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Chassis and vector

The part was inserted into pET29b vector downstream a T7 promoter and lac operon. Thus, the expression of the operon was induced using Isopropyl β-D-1-thiogalactopyranoside (IPTG). E. coli (BL21) was chosen as the chassis because of its ability in better protein expression. After transforming the bacteria with plasmid, colonies were selected and screened for presence of plasmid. A successful colony was then selected for experiments.

Experiment

Glucose (Positive control)	pET29B in BL21 (Negative 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	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	Pure VFAs
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	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