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Team:Macquarie Australia/Protocols/HO
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| - | <h1><center>Heme Oxygenase Characterisation</center></h1> | + | <h1><center>Heme Oxygenase Function Characterisation</center></h1> |
<h2>Rationale</h2> | <h2>Rationale</h2> | ||
<p>To determine if our heme oxygenase BioBrick part was functional we needed to stimulate the pathway. We could easily determined if heme oxygenase was functional due to the production of the pigment biliverdine. As our BioBrick was under control of the T7 promoter, the enzyme would need to be induced using IPTG. The heme pathway was also stimulated using d-aminolevulinic acid (ALA). If our Heme oxygenase was functional than the cells would be green in appearance.</p> | <p>To determine if our heme oxygenase BioBrick part was functional we needed to stimulate the pathway. We could easily determined if heme oxygenase was functional due to the production of the pigment biliverdine. As our BioBrick was under control of the T7 promoter, the enzyme would need to be induced using IPTG. The heme pathway was also stimulated using d-aminolevulinic acid (ALA). If our Heme oxygenase was functional than the cells would be green in appearance.</p> | ||
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<h2>Protocol</h2> | <h2>Protocol</h2> | ||
<blockquote><ol> | <blockquote><ol> | ||
| - | <li>After transforming BL21 <i>E. coli</i> with the BioBrick, LB media (2 mL) was inoculated and incubated at 37°C until the culture | + | <li>After transforming BL21 <i>E. coli</i> with the BioBrick, LB media (2 mL) was inoculated and incubated at 37°C until the culture had reached an OD<sub>600</sub>=0.50 .</li> |
<li>ALA (20 µL, Final concentration 1 mM) was added and the cultures were incubate at room temperature for 30 minutes.</li> | <li>ALA (20 µL, Final concentration 1 mM) was added and the cultures were incubate at room temperature for 30 minutes.</li> | ||
<li>IPTG (20 µL, Final concentration 1 mM) was added and they were left to incubate overnight.</li> | <li>IPTG (20 µL, Final concentration 1 mM) was added and they were left to incubate overnight.</li> | ||
| - | <li>The cells were then spun down, the supernatant poured off and the | + | <li>The cells were then spun down, the supernatant poured off and the pigment observed.</li> |
</ol></blockquote> | </ol></blockquote> | ||
Latest revision as of 22:10, 25 September 2012
Heme Oxygenase Function Characterisation
Rationale
To determine if our heme oxygenase BioBrick part was functional we needed to stimulate the pathway. We could easily determined if heme oxygenase was functional due to the production of the pigment biliverdine. As our BioBrick was under control of the T7 promoter, the enzyme would need to be induced using IPTG. The heme pathway was also stimulated using d-aminolevulinic acid (ALA). If our Heme oxygenase was functional than the cells would be green in appearance.
Protocol
- After transforming BL21 E. coli with the BioBrick, LB media (2 mL) was inoculated and incubated at 37°C until the culture had reached an OD600=0.50 .
- ALA (20 µL, Final concentration 1 mM) was added and the cultures were incubate at room temperature for 30 minutes.
- IPTG (20 µL, Final concentration 1 mM) was added and they were left to incubate overnight.
- The cells were then spun down, the supernatant poured off and the pigment observed.
