Materials & Methods

If you choose to create a model during your project, please write about it here. Modeling is not an essential part of iGEM, but we encourage any and all teams to model some aspect of their project. See previous "Best Model" awards for more information.

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Materials

Reagents

• 20 mM KH2PO4 and 10 mM KCl (pH 8) solution
• 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution
• Biomass recovered from the 40 mL medium
• Fresh Laemmli Buffer
• Lysozyme (20 mg/mL)

Equipment

• Centrifuge
• Micropipettes
• Conical tubes
• Centrifugal tubes

Procedure

1. Centrifuge 40 mL medium 5000 rpm, 30 min. Remove supernatant.
2. Resuspend pellet in 4 mL of a cold 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution.
3. Add 70uL of Lysozyme.
4. Incubate in shaker 200 rpm the biomass-lysozyme solution at 37° C for 60 minutes.
5. Centrifuge cell lysate at 14,000 rpm for 10 minutes. If the supernatant is not crystalline, the sample has to be centrifuged again. Store soluble phase at 4°C in a conical tube, covered with aluminum for chromatography.
6. Take 20 µL of the supernatant and mix with 20 µL of Laemmli buffer for SDS PAGE analysis.
7. The insoluble phase obtained from the previous centrifugation has to be washed twice with a 1% SDS solution and 20 mM KH2PO4 and 10 mM KCl (pH 8) solution. Then, mix insoluble phase with 20ul Laemmli buffer for SDS PAGE analysis.
8. Run a SDS PAGE with iGEM ITESM CEM’s protocol.

*Do not cease to contemplate that all samples should be perfectly mixed with the indicated reagents (Laemmli Buffer) before they are boiled for 5 minutes. In the case of the inclusion bodies, the boiling should be carried out for 10 minutes.

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