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Team:Macquarie Australia/WetLab/Protocols/Operons
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| + | <ul style="list-style-type: decimal;"> | ||
| + | <li>Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol. | ||
| + | <li>Screen plates for transformant colonies and place in 5mL of LB broth with appropriate antibiotic concentration at 37<sup>o</sup>C with shaking until OD600¬ is 2.0 – 3.0.</li> | ||
| + | <li>Take whole volume and place in 45mL of LB broth in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37<sup>o</sup>C with shaking until OD600 is 0.5-0.7.</li> | ||
| + | <li>Transfer 1M IPTG in a 1:1000 dilution into flask. Incubate at 15<sup>o</sup>C with shaking overnight. </li> | ||
| + | <li>Cell pellets were collected through centrifugation at 12000 rpm for 5 mins. </li> | ||
| + | <li>Cell pellets were resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl<sub>2</sub>, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate.</li> | ||
| + | </ul> | ||
| + | |||
| + | <h4>Operon 1 functional assay</h4> | ||
| + | <ul style="list-style-type: decimal;"> | ||
| + | <li>Collect whole lysate from induced operon 1 containing ChlI1 & ChlD expressed proteins, and ChlI<sub>2</sub> expressed protein only as a negative control. Prepare and map out 384-well microtitre plate for assay.</li> | ||
| + | <li>Prepare master mix of recombinant ChlH (500nM), GUN4 (500nM) & Protoporphyrin IX (1µM) to cover sufficient amount of expressed operon wells, and negative control wells to be assayed (25µl/well).</li> | ||
| + | <li>Prepare master mix of recombinant ChlD (500nM) and ChlI1 (500nM) to cover sufficient amount of positive control wells to be assayed (10µl/well).</li> | ||
| + | <li>Prepare assay buffer containing: MgCl<sub>2</sub> (14mM), ATP (4mM), DDT (1mM), Trycine/NaOH (50mM; pH 8.0), 10% glycerol.</li> | ||
| + | <li>Operon assay wells: 10uL I1/D lysate // 15uL buffer // 25uL purified H/GUN4/PROTO master.</li> | ||
| + | <li>Positive Control assay wells: 10uL recombinant I1/D master // 15uL buffer // 25uL H/GUN4/PROTO master.</li> | ||
| + | <li>Negative Control assay wells: 2.5uL ChlI2 lysate // 10uL master // H/GUN4/PROTO master.</li> | ||
| + | <li>Place microtitre tray in Pherastar FS. Measure emission at 595nm with excitation 420nm, cycling every 30s for 1h.</li> | ||
| + | <li>Following emission measurement, measure fluorescence spectra from 550-660nm with 420nm excitation to identify Mg-Protoporphyrin IX formation (595nm) and Protoporphyin IX presence (635nm).</li> | ||
| + | </ul> | ||
</div> | </div> | ||
Revision as of 13:19, 17 October 2014
- Re-transform restriction-digest screened plasmids according to the heat shock transformation protocol.
- Screen plates for transformant colonies and place in 5mL of LB broth with appropriate antibiotic concentration at 37oC with shaking until OD600¬ is 2.0 – 3.0.
- Take whole volume and place in 45mL of LB broth in a sterile conical flask containing appropriate antibiotic concentration. Incubate at 37oC with shaking until OD600 is 0.5-0.7.
- Transfer 1M IPTG in a 1:1000 dilution into flask. Incubate at 15oC with shaking overnight.
- Cell pellets were collected through centrifugation at 12000 rpm for 5 mins.
- Cell pellets were resuspended in re-suspension buffer (Containing: 10% glycerol w/v, 50mM Tricene NaOH pH 8.0, 2mM MgCl2, 1mM DTT) and whole lysate was collected through a French Press for functional assays of operon protein products in lysate.
Operon 1 functional assay
- Collect whole lysate from induced operon 1 containing ChlI1 & ChlD expressed proteins, and ChlI2 expressed protein only as a negative control. Prepare and map out 384-well microtitre plate for assay.
- Prepare master mix of recombinant ChlH (500nM), GUN4 (500nM) & Protoporphyrin IX (1µM) to cover sufficient amount of expressed operon wells, and negative control wells to be assayed (25µl/well).
- Prepare master mix of recombinant ChlD (500nM) and ChlI1 (500nM) to cover sufficient amount of positive control wells to be assayed (10µl/well).
- Prepare assay buffer containing: MgCl2 (14mM), ATP (4mM), DDT (1mM), Trycine/NaOH (50mM; pH 8.0), 10% glycerol.
- Operon assay wells: 10uL I1/D lysate // 15uL buffer // 25uL purified H/GUN4/PROTO master.
- Positive Control assay wells: 10uL recombinant I1/D master // 15uL buffer // 25uL H/GUN4/PROTO master.
- Negative Control assay wells: 2.5uL ChlI2 lysate // 10uL master // H/GUN4/PROTO master.
- Place microtitre tray in Pherastar FS. Measure emission at 595nm with excitation 420nm, cycling every 30s for 1h.
- Following emission measurement, measure fluorescence spectra from 550-660nm with 420nm excitation to identify Mg-Protoporphyrin IX formation (595nm) and Protoporphyin IX presence (635nm).
