Line 46: | Line 46: | ||
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 OD<sub>600</sub>. The composition of each of the replicate in the flasks is shown below: | 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 OD<sub>600</sub>. The composition of each of the replicate in the flasks is shown below: | ||
− | < | + | <h6>pET29B in BL21 (Negative control)</h6> |
− | <ul> 10 ml O/Ns in LB+Kan</ | + | <ul> |
− | < | + | <li> 10 ml O/Ns in LB+Kan</li> |
− | < | + | <li> "Syn poo" fermented supernatant {..!!hyperlink to eng page for syn poo protocol..}</li> |
− | < | + | <li> 100 uL MgSO<sub>4</sub></li> |
− | < | + | <li> 5 uL CaCl<sub>2</sub></li> |
+ | <li> 10 ml M9 salts</li> | ||
+ | <li> 5 uL 1M IPTG</li> | ||
+ | </ul> | ||
+ | |||
+ | {..........what else was added was supernatant added....} | ||
<h5>Glucose (Positive control)</h5> | <h5>Glucose (Positive control)</h5> | ||
− | <ul> 10 ml O/Ns in LB+Kan</ | + | <ul> |
− | < | + | <li> 10 ml O/Ns in LB+Kan</li> |
− | < | + | <li> 7 ml 20% Glucose {..!!cite why this conc chosen..}</li> |
− | < | + | <li> 100 uL MgSO<sub>4</sub></li> |
− | < | + | <li> 5 uL CaCl<sub>2</sub></li> |
+ | <li> 10 ml M9 salts</li> | ||
+ | <li> 5 uL 1M IPTG</li> | ||
+ | <li> 20 ml dH<sub>2</sub>O</li> | ||
+ | </ul> | ||
<h5>Fermented "syn poo" supernatant</h5> | <h5>Fermented "syn poo" supernatant</h5> | ||
− | <ul> 10 ml O/Ns in LB+Kan</ | + | <ul> |
− | < | + | <li> 10 ml O/Ns in LB+Kan</li> |
− | < | + | <li> 10 ml "Syn poo" fermented supernatant {..!!hyperlink to eng page for syn poo protocol..}</li> |
− | < | + | <li> 100 uL MgSO<sub>4</sub></li> |
− | < | + | <li> 5 uL CaCl<sub>2</sub></li> |
+ | <li> 10 ml M9 salts</li> | ||
+ | <li> 5 uL 1M IPTG</li> | ||
+ | <li> 23 ml dH<sub>2</sub>O</li> | ||
+ | </ul> | ||
<h5>Pure VFAs</h5> | <h5>Pure VFAs</h5> | ||
− | <ul> 10 ml O/Ns in LB+Kan</ | + | <ul> |
− | < | + | <li> 10 ml O/Ns in LB+Kan</li> |
− | < | + | <li> VFAs</li> |
− | < | + | <li> 410 uL propionic acid</li> |
− | < | + | <li> 118 uL acetic acid</li> |
− | < | + | <li> 55 uL butyric acid</li> |
− | < | + | <li> 100 uL MgSO<sub>4</sub></li> |
− | < | + | <li> 5 uL CaCl<sub>2</sub></li> |
+ | <li> 10 ml M9 salts</li> | ||
+ | <li> 5 uL 1M IPTG</li> | ||
+ | <li> 30 ml dH<sub>2</sub>O</li> | ||
+ | </ul> | ||
<p> The OD<sub>600</sub> readings of .....???? were taken and recorded: | <p> The OD<sub>600</sub> readings of .....???? were taken and recorded: |
Revision as of 05:30, 21 October 2017
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
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
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 |