Difference between revisions of "Team:Calgary/BetaOxidation"

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     <th><b>Glucose (Positive control)</b></th></tr>
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     <th><b>Glucose (Positive control)</b></th>
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    <th><b>pET29B in BL21 (Negative control)</b></th>
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            <b>pET29B in BL21 (Negative control)</b></th>
 
 
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   <tr><th><b>Fermented "syn poo" supernatant</b></th></tr>
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   <tr><th><b>Fermented "syn poo" supernatant</b></th>
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    <th><b>Pure VFAs</b></th></tr>
 
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             <ul>
 
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             <li> 10 ml O/Ns in LB+Kan</li>
 
             <li> 10 ml O/Ns in LB+Kan</li>

Revision as of 00:13, 24 October 2017

Header

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) 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