Team:Macquarie Australia/WetLab/Protocols/Operons

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<div class="cont-out">
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<h3>Functional Assays</h3><br/>
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<h4>Induction of Operons</h4>
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<ul style="list-style-type: decimal;">
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<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>
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<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>
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<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>
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<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>
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</ul><br/>
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<h4>Operon 1 functional assay</h4>
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<ul style="list-style-type: decimal;">
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<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>
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<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>
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<li>Prepare assay buffer containing: MgCl<sub>2</sub> (14mM), ATP (4mM), DDT (1mM), Trycine/NaOH (50mM; pH 8.0), 10% glycerol.</li>
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<li>Operon assay wells: 10uL I1/D lysate // 15uL buffer // 25uL purified H/GUN4/PROTO master.</li>
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<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 ChlI<sub>2</sub> 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>
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<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>
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</ul><br/>
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<h4>UHPLC protocol</h4>
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<p>The UHPLC protocol was performed according to Benton et. al. 2012. Briefly, methanol extracted compounds were separated on a reverse phase C18 column. Compounds were detected at an excitation wavelength of 404nm and emission wavelength of 618 nm.</p>
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<h3>References</h3>
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<p><ul style="list-style-type: decimal;">
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<li>Benton, C. M., Lim, C. K., Moniz, C., & Jones, D. J. (2012). Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation. Biomed Chromatogr, 26(6), 714-719. doi: 10.1002/bmc.1720</li>
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</ul></p>
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<h3></h3>
</div>
</div>

Latest revision as of 02:38, 18 October 2014

Protocols


Functional Assays


Induction of Operons

  • 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).

UHPLC protocol

The UHPLC protocol was performed according to Benton et. al. 2012. Briefly, methanol extracted compounds were separated on a reverse phase C18 column. Compounds were detected at an excitation wavelength of 404nm and emission wavelength of 618 nm.

References

  • Benton, C. M., Lim, C. K., Moniz, C., & Jones, D. J. (2012). Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation. Biomed Chromatogr, 26(6), 714-719. doi: 10.1002/bmc.1720

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