Difference between revisions of "Team:Harvard/Protocols"

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<h5>Cloning</h5>
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      <h4 class="panel-title">
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        <a data-toggle="collapse" data-parent="#accordion" href="#collapse1">Congo Red Agar Plates</a>
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      <div class="panel-body">
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        <h5>Purpose</h5>
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        <p>To visualize curli production in curli-producing colonies with fluorescence imaging</p>
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        <h5>Materials</h5>
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        <ul>
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          <li>10 g agar</li>
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          <li>12.5 g LB</li>
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          <li>dH2O</li>
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          <li>10 g agar</li>
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          <li>96 well plate</li>
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        </ul>
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        <h5>Methods</h5>
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        <ol>
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          <li>Added 10 g agar and 12.5 g LB to 500 mL of Milli-Q water</li>
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          <li>Autoclaved for 30 minutes under “liquids” setting</li>
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          <li>Put on bench until no longer scalding to the touch</li>
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          <li>Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.</li>
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          <li>Added Congo Red to a final concentration of 0.0025 g per 100 mL, arabinose to a final concentration of 100 μM, and kanamycin to a final concentration of 50 μg/mL
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              <ul>
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                <li>If using a different inducible promoter, adjust inducer accordingly</li>
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                <li>If using a different selection marker, adjust antibiotic accordingly</li>
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              </ul>
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          </li>
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          <li>Mix thoroughly</li>
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          <li>Pour immediately into plates</li>
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          <li>Pop superficial air bubbles with a Bunsen burner</li>
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          <li>Let dry on bench for 2-3 hours with lids ajar</li>
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        </ol>
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      <p> Recipe adapted from Peter Nguyen et al. as detailed in <a href="https://www.nature.com/articles/ncomms5945">“Programmable Biofilm-Based Materials from Engineered Curli Nanofibres”</a> from <i>Nature Communications</i></p>
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<h5>Protein Expression Measurements</h5>
 
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           <li>96 well plate</li>
 
           <li>96 well plate</li>
 
         </ul>
 
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        <h5>Methods</h5>
 
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        <ol>
 
 
         <h5>Methods</h5>
 
         <h5>Methods</h5>
 
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Revision as of 03:29, 30 October 2017

Harvard OPTI POLY

Protocols


Cloning

Congo Red Agar Plates

Purpose

To visualize curli production in curli-producing colonies with fluorescence imaging

Materials
  • 10 g agar
  • 12.5 g LB
  • dH2O
  • 10 g agar
  • 96 well plate
Methods
  1. Added 10 g agar and 12.5 g LB to 500 mL of Milli-Q water
  2. Autoclaved for 30 minutes under “liquids” setting
  3. Put on bench until no longer scalding to the touch
  4. Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
  5. Added Congo Red to a final concentration of 0.0025 g per 100 mL, arabinose to a final concentration of 100 μM, and kanamycin to a final concentration of 50 μg/mL
    • If using a different inducible promoter, adjust inducer accordingly
    • If using a different selection marker, adjust antibiotic accordingly
  6. Mix thoroughly
  7. Pour immediately into plates
  8. Pop superficial air bubbles with a Bunsen burner
  9. Let dry on bench for 2-3 hours with lids ajar

Recipe adapted from Peter Nguyen et al. as detailed in “Programmable Biofilm-Based Materials from Engineered Curli Nanofibres” from Nature Communications

Protein Expression Measurements

Congo Red Spin Down Assay

Materials
  • 1 mL LUDOX
  • H2O
  • 96 well plate
Methods
  1. Induce expression of curli by adding 1M IPTG to liquid culture, to a final concentration of 250 µM. For example, if you have 3 mL culture, add 0.75 µL of 1M IPTG solution.
  2. Incubate liquid culture overnight for optimal curli expression.
  3. Make 0.015% Congo Red solution by diluting 1% congo red solution. For example, 150 µl of 1% congo red solution in 10 ml DI water.
  4. Remove the culture from the 25C incubator. Add 1 ml of the culture into a cleaned 2ml Eppendorf tube.
  5. Pellet the cells by centrifuge at 6800 g (8000 rpm) at room temperature for 10 minutes.
  6. Remove the supernatant by decanting, and using a pipette to siphon off as much of the supernatant without disturbing the pellet.
  7. Resuspend cells in 1 mL of PBS gently by pipetting up and down with a pipette.
  8. Add 100 µl of 0.015% congo red solution to the tube. Mix gently by pipetting. For the control tube, add the congo red solution to 1 ml of pure PBS.
  9. Leave the solution at room temperature for 10 minutes
  10. Pellet by centrifuging at 14,000 rpm at room temperature for 10 minutes.
  11. Add 150 µl of the supernatant to a well of 96-well plate along with pure PBS, and control PBS-Congo Red.
  12. Using a plate reader, measure absorbance at wavelength 490 nm.

Protocol Courtesy of Bom Praveschotinunt of Wyss Institute, Joshi Lab