Team:Imperial College/Protocols

From 2013.igem.org

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<h1>Protocols</h1>
 
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<b>Glucose Assay</b>  
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<h1>Materials</h1>
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<b>Waste Conditioned Media (WCM)</b>
<b>Waste Conditioned Media (WCM)</b>
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<p>We took 6g [http://en.wikipedia.org/wiki/Refuse-derived_fuel SRF] (Solid Recovered Fuel) and to this added 300 mL LB. In addition to this we prepared 300 mL LB. Post-autoclave, the SRF LB was sterile filtrated into 50 mL falcons, which we will now call Waste Conditioned Media (WCM). To both the LB and WCM, 150 µl of chloramphenicol was added. 100 mL WCM was added to 3 pre-sterilised flasks, this was repeated for LB.</p>
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<p>We added 1g[http://en.wikipedia.org/wiki/Refuse-derived_fuel SRF] (Solid Recovered Fuel)/50 mL LB and then autoclaved the mixture. Once autoclaved, large waste chunks were removed through filter sterilisation (0.2uM filters) before being used in growth assays as waste conditioned media (WCM).
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<p>1mL stress response transformed (BBa_K639003) MG1655 grown overnight was added to each of the flasks. This was placed on a shaking incubator at 37°C. T=0 was taken after an hour for the bacteria to normalise to their environment. After this time points were taken every 6 or 12h. </p>
 
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<b>[http://biolabs.tmcc.edu/Micro%20Web/SerialDilutions.pdf Serial Dilution]</b>
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<h1>Protocols and Assays</h1>
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<b>Preparation of Poly DEGA plates.</b>
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<b>Waste Conditioned Assay</b>  
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<p>In addition to LB, place 0.5% poly DEGA in 20 mM Tris-HCl at pH 8.0 into the solution. In a 300 mL solution this translates to 1.5 mL poly DEGA. As poly DEGA is very viscous, before addition, ensure LB agar is kept at 70°C. We then used a magnetic stirrer to ensure that the poly DEGA was thoroughly mixed.</p>
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O/N cultures of MG1655 transformed with (BBa_K639003) were diluted to OD 0.05 in either fresh LB or waste conditioned media and plated into 96 well plates (200ul/well). OD600 was read at the indicated time points and media only (LB) was taken away as background signal.
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<br> <br>
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<b>[http://biolabs.tmcc.edu/Micro%20Web/SerialDilutions.pdf Serial Dilution]</b>
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<br> <br>
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<b>Preparation of Poly DEGA plates.</b>
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<p>In addition to LB, place 0.5% poly DEGA in 20 mM Tris-HCl at pH 8.0 into the solution. In a 300 mL solution this translates to 1.5 mL poly DEGA. As poly DEGA is very viscous, before addition, ensure LB agar is kept at 70°C. We then used a magnetic stirrer to ensure that the poly DEGA was thoroughly mixed.</p>
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Revision as of 18:32, 27 August 2013

Materials

Waste Conditioned Media (WCM)

We added 1g[http://en.wikipedia.org/wiki/Refuse-derived_fuel SRF] (Solid Recovered Fuel)/50 mL LB and then autoclaved the mixture. Once autoclaved, large waste chunks were removed through filter sterilisation (0.2uM filters) before being used in growth assays as waste conditioned media (WCM).

Protocols and Assays



Waste Conditioned Assay

O/N cultures of MG1655 transformed with (BBa_K639003) were diluted to OD 0.05 in either fresh LB or waste conditioned media and plated into 96 well plates (200ul/well). OD600 was read at the indicated time points and media only (LB) was taken away as background signal.



[http://biolabs.tmcc.edu/Micro%20Web/SerialDilutions.pdf Serial Dilution]

Preparation of Poly DEGA plates.

<p>In addition to LB, place 0.5% poly DEGA in 20 mM Tris-HCl at pH 8.0 into the solution. In a 300 mL solution this translates to 1.5 mL poly DEGA. As poly DEGA is very viscous, before addition, ensure LB agar is kept at 70°C. We then used a magnetic stirrer to ensure that the poly DEGA was thoroughly mixed.


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