Team:ITESM-CEM/Project/Materials

From 2014.igem.org

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       <sub2><a href="#Four" style="color: #FFF;">Four</a></sub2>
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       <sub2><a href="#Four" style="color: #FFF;">Protein Expression</a></sub2>
       <sub2><a href="#Five" style="color: #FFF;">Five</a></sub2>
       <sub2><a href="#Five" style="color: #FFF;">Five</a></sub2>
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<a name="Four"><h2>Experiment Four</h2></a>  
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<a name="Four"><h2>Recombinant Protein Expression Protocol</h2></a>
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<h4>Materials</h4>  
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       <p style="text-align: justify; text-justify: inter-word;">
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    Reagents<br><br>
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•  20 mM KH2PO4 and 10 mM KCl (pH 8) solution<br>
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•  300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution<br>
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•  Biomass recovered from the 40 mL medium<br>
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•  Fresh Laemmli Buffer<br>
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•  Lysozyme (20 mg/mL)<br><br>
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Equipment<br><br>
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•  Centrifuge<br>
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•  Micropipettes<br>
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•  Conical tubes<br>
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•  Centrifugal tubes<br><br>
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<h4>Procedure</h4> <br><br>
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1. Centrifuge 40 mL medium 5000 rpm, 30 min. Remove supernatant.<br>
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2. Resuspend  pellet in 4 mL of a cold 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution.<br>
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3. Add 70uL of Lysozyme.<br>
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4. Incubate in shaker 200 rpm the biomass-lysozyme solution at 37° C for 60 minutes.<br>
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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.<br>
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6. Take 20 µL of the supernatant and mix with 20 µL of Laemmli buffer for SDS PAGE analysis.<br>
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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.<br>
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8. Run a SDS PAGE with iGEM ITESM CEM’s protocol.<br></p>
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<p style="text-align: justify; text-justify: inter-word;">
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*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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</p>
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<gotop><a href="#top">Back to top ↑</a></gotop><br><br>

Revision as of 18:02, 17 October 2014

TEC-CEM | Project

ITESM-CEM | Enzy7-K me

Project 3014
 

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.

Experiment One

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Vivamus fringilla porta nisi sed dictum. Aliquam at rutrum nisl. Ut eros quam, condimentum sed pretium et, euismod vitae tellus. Curabitur eu blandit massa. Vestibulum at euismod purus. Sed quis pretium ligula. Cras non tristique nulla. Nunc finibus risus non purus malesuada viverra. Cras bibendum augue eu lorem faucibus, nec auctor mi iaculis. Vivamus sagittis, mi non gravida accumsan, odio dui pellentesque leo, a varius dui mauris eu diam. Maecenas laoreet tellus sed porta efficitur.

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Experiment Two

Etiam tempus mi pulvinar purus iaculis bibendum. Vivamus vel risus eu enim volutpat finibus. Nullam bibendum est sit amet arcu lobortis, id laoreet ex vestibulum. Curabitur fringilla eleifend lacus, nec ornare nibh imperdiet sed. Maecenas vel velit consectetur, tempus libero quis, consectetur ex. Nulla porttitor pharetra velit. Curabitur tristique, dolor ut sodales euismod, diam diam ultricies arcu, at tincidunt tellus neque in felis. Etiam ut tempor ligula. Sed at dui sapien.

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Experiment Three

Proin aliquam nibh id elementum pellentesque. Suspendisse mollis est ut felis sagittis mollis. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Etiam accumsan ex ante, quis lobortis erat fermentum ac. Sed et egestas libero. Donec id diam vitae leo consequat interdum. Ut in sem in quam pretium finibus vitae non lectus.

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Recombinant Protein Expression Protocol

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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Experiment Five

Praesent molestie consectetur nunc eu gravida. Praesent imperdiet quam vitae erat dapibus sagittis vitae id libero. Etiam vitae tortor massa. Donec vehicula lacus non sem eleifend, sed commodo nunc lobortis. Nulla vitae volutpat mi. Donec vestibulum ultrices lectus, in venenatis elit luctus sit amet. Aliquam eget lacus accumsan, dictum dolor accumsan, venenatis lectus. Ut eget mi facilisis, accumsan odio ac, viverra ipsum. Integer efficitur massa sit amet cursus ultrices. Etiam porta, nisl et tristique egestas, lectus augue convallis nulla, ut ultrices odio dui nec lectus. Morbi facilisis tortor aliquet sagittis auctor. Vestibulum ante ipsum primis in faucibus orci luctus et ultrices posuere cubilia Curae; Etiam tincidunt aliquet sem id lobortis. Nam tincidunt ultrices lorem dictum pretium.

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