Difference between revisions of "Team:Imperial College/Experiments"
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<div type="button" class="accordion"> Description </div> | <div type="button" class="accordion"> Description </div> | ||
<div class="description"> | <div class="description"> | ||
| − | <p> | + | <p>This protocol describes how to measure E. coli growth and GFP expression over time with a plate reader, using the optical density at 600 nm as signal for cell density and excitation and emission wavelengths of 475. |
| + | All characterization was performed using the FLUOstar BMG Labtech in greiner bioone F-bottom dark plates with clear bottom, to decrease fluorescence background. | ||
| + | Each cycle of this experiment takes two days and has to be repeated two days, in order to have 2 biological and 2 technical replicates, for a total of 4 days and 4 96-well plates used. | ||
| + | </p> | ||
</div> | </div> | ||
| − | <div type="button" class="accordion"> | + | <div type="button" class="accordion">Materials</div> |
<div class="panel expt2p"> | <div class="panel expt2p"> | ||
| − | + | <ul> | |
| + | <li>Autoclaved LB media</li> | ||
| + | <li>Autoclaved 10x concentated LB media</li> | ||
| + | <li>500 uM pyocyanin stock solution (from Sigma-Aldrich, CAS: No. 86-66-5)</li> | ||
| + | <li>1250 mM potassium ferricyanide stock solution (from Sigma-Aldrich, CAS: No. 13746-66-2)</li> | ||
| + | <li>500 mM ferrocyanide stock solution (from Sigma-Aldrich, CAS 14459-95-1 )</li> | ||
| + | <li>2% Sodium Sulfite Solution</li> | ||
| + | <li> | ||
| + | Agar plates with colonies of E. coli DJ901 strains with respective constructs: PixCell Patterning Circuit 1 (BBa_K2862021). PixCell Patterning Circuit 2 (BBa_K2862022), negative control (DJ901 WT) and positive control (constitutively expressed GFP).</li> | ||
| + | <li>37°C shaking incubator</li> | ||
| + | <li>BMG Labtech FLUOstar plate reader</li> | ||
| + | <li>4x Greiner BioOne 96-F 96-well microplate (black with clear bottom)</li> | ||
| + | <li>14 mL culture tubes</li> | ||
| + | <li>autoclaved eppendorfs</li> | ||
| + | </ul> | ||
</div> | </div> | ||
| − | <div type="button" class="accordion"> | + | <div type="button" class="accordion">Notes</div> |
<div class="panel expt2p"> | <div class="panel expt2p"> | ||
| − | <p> | + | <p>Work in very sterile conditions. To prevent contaminations in the blanks, use double antibiotics. |
| − | + | </br> | |
| − | + | Store pyocyanin and ferrocyanide and ferricyanide solutions in the freezer until use. | |
| + | </br> | ||
| + | We used 10x concentrated LB in order not to dilute nutrients at higher pyocyanin concentration; this was back diluted with autoclaved water. | ||
| + | |||
| + | </p> | ||
</div> | </div> | ||
| − | <div type="button" class="accordion" > | + | <div type="button" class="accordion" >Procedure</div> |
<div class="panel expt2p"> | <div class="panel expt2p"> | ||
<p>Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p> | <p>Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.</p> | ||
| + | <ol> | ||
| + | <li>Grow starter liquid cultures of cells (Day 0, 10-15 min preparation + overnight)</li> | ||
| + | |||
| + | <p>Working in very sterile conditions, take 5 14mL culture tubes and prepare them according to table below: </p> | ||
| + | |||
| + | <table class="tg"> | ||
| + | <tr> | ||
| + | <th class="tg-1wig">Label</th> | ||
| + | <th class="tg-1wig"><span style="font-style:italic">E. coli</span> strain</th> | ||
| + | <th class="tg-1wig">Construct</th> | ||
| + | <th class="tg-1wig">Antibiotic (Ab)</th> | ||
| + | <th class="tg-1wig">V Ab (uL)</th> | ||
| + | <th class="tg-1wig">V LB (mL)</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="tg-0lax">PC1</td> | ||
| + | <td class="tg-0lax">DJ901</td> | ||
| + | <td class="tg-0lax">BBa_K2862021</td> | ||
| + | <td class="tg-096r">Kan + Amp</td> | ||
| + | <td class="tg-0lax">5 + 5</td> | ||
| + | <td class="tg-0lax">5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="tg-0lax">PC2</td> | ||
| + | <td class="tg-0lax">DJ901</td> | ||
| + | <td class="tg-0lax">BBa_K2862022</td> | ||
| + | <td class="tg-0lax">Kan + Amp</td> | ||
| + | <td class="tg-0lax">5 + 5</td> | ||
| + | <td class="tg-0lax">5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="tg-0lax">-</td> | ||
| + | <td class="tg-0lax">DJ901</td> | ||
| + | <td class="tg-0lax">none</td> | ||
| + | <td class="tg-0lax">Kan</td> | ||
| + | <td class="tg-0lax">5 </td> | ||
| + | <td class="tg-0lax">5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="tg-0lax">+</td> | ||
| + | <td class="tg-0lax">DJ901</td> | ||
| + | <td class="tg-0lax">GFP_Boo</td> | ||
| + | <td class="tg-0lax">Kan + Chl</td> | ||
| + | <td class="tg-0lax">5 + 5</td> | ||
| + | <td class="tg-0lax">5</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td class="tg-0lax">Blanks</td> | ||
| + | <td class="tg-0lax">None</td> | ||
| + | <td class="tg-0lax">None</td> | ||
| + | <td class="tg-0lax">Kan + Amp + Chl</td> | ||
| + | <td class="tg-0lax">5 + 5</td> | ||
| + | <td class="tg-0lax">5</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
</div> | </div> | ||
<div class="clr"></div> | <div class="clr"></div> | ||
Revision as of 15:03, 16 October 2018
Methods
Contents
- Assembling the Pixcell Constructs
- Characterizing Pixcell Constructs
- Electrochemistry of Redox Modulators
- Spatial Electronic Control of Gene Induction
- Creating the Sox Library
- Characterising the Sox Library
- Testing Phenazine Methosulfate
- Biocontainment- Gp2 Growth Inhibition
- Fabric Bioprinting - Melanin
Experiments
Assembling the Pixcell Constructs
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Characterizing Pixcell Constructs
This protocol describes how to measure E. coli growth and GFP expression over time with a plate reader, using the optical density at 600 nm as signal for cell density and excitation and emission wavelengths of 475. All characterization was performed using the FLUOstar BMG Labtech in greiner bioone F-bottom dark plates with clear bottom, to decrease fluorescence background. Each cycle of this experiment takes two days and has to be repeated two days, in order to have 2 biological and 2 technical replicates, for a total of 4 days and 4 96-well plates used.
- Autoclaved LB media
- Autoclaved 10x concentated LB media
- 500 uM pyocyanin stock solution (from Sigma-Aldrich, CAS: No. 86-66-5)
- 1250 mM potassium ferricyanide stock solution (from Sigma-Aldrich, CAS: No. 13746-66-2)
- 500 mM ferrocyanide stock solution (from Sigma-Aldrich, CAS 14459-95-1 )
- 2% Sodium Sulfite Solution
- Agar plates with colonies of E. coli DJ901 strains with respective constructs: PixCell Patterning Circuit 1 (BBa_K2862021). PixCell Patterning Circuit 2 (BBa_K2862022), negative control (DJ901 WT) and positive control (constitutively expressed GFP).
- 37°C shaking incubator
- BMG Labtech FLUOstar plate reader
- 4x Greiner BioOne 96-F 96-well microplate (black with clear bottom)
- 14 mL culture tubes
- autoclaved eppendorfs
Work in very sterile conditions. To prevent contaminations in the blanks, use double antibiotics. Store pyocyanin and ferrocyanide and ferricyanide solutions in the freezer until use. We used 10x concentrated LB in order not to dilute nutrients at higher pyocyanin concentration; this was back diluted with autoclaved water.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
- Grow starter liquid cultures of cells (Day 0, 10-15 min preparation + overnight)
Working in very sterile conditions, take 5 14mL culture tubes and prepare them according to table below:
| Label | E. coli strain | Construct | Antibiotic (Ab) | V Ab (uL) | V LB (mL) |
|---|---|---|---|---|---|
| PC1 | DJ901 | BBa_K2862021 | Kan + Amp | 5 + 5 | 5 |
| PC2 | DJ901 | BBa_K2862022 | Kan + Amp | 5 + 5 | 5 |
| - | DJ901 | none | Kan | 5 | 5 |
| + | DJ901 | GFP_Boo | Kan + Chl | 5 + 5 | 5 |
| Blanks | None | None | Kan + Amp + Chl | 5 + 5 | 5 |
Electrochemistry of Redox Modulators
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Spatial Electronic Control of Gene Induction
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Creating the Sox Library
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Characterising the Sox Library
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Testing Phenazine Methosulfate
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Biocontainment- Gp2 Growth Inhibition
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Fabric Bioprinting - Melanin
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Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat. Lorem ipsum dolor, sit amet consectetur adipisicing elit. Competant cell protocol Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
Lorem ipsum dolor, sit amet Heatshock protocol consectetur adipisicing elit. Recusandae dolore, tenetur expedita in architecto, repellat rerum minima cumque exercitationem debitis laborum fugit aliquam quo molestiae. Alias eius nostrum aspernatur quaerat.
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