Notebook:Federico Castro M/2008/01/12

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Proposed Procedure

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• Status: Pending for aproval
• Issues: None

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I want to recover 2 biobricks from the kit plates in order assemble the Repressilator. This will improve my skills and the knowledge of how oscillators work. The biobrick <bbpart>BBa_S03151</bbpart> is a devise compose by 3 inverters and if joined with <bbpart>BBa_E0430</bbpart> it will form the repressilator. <bbpart>BBa_E0430</bbpart> eill be usefull to assemble the diffusible signal oscillator.

The whole procedure should be easy cheap and educative. I await for approval for this procedure.

Biobrick to be extracted

• <bbpart>BBa_S03151</bbpart> available at well 6L at iGEM 2007 Parts Kit Plate 1 in plasmid pSB4A3
• <bbpart>BBa_E0430</bbpart> available at well 11A at iGEM 2007 Parts Kit Plate 1 in plasmid pSB1A2

You can see the information about this biobricks here [1]

The following protocol is proposed for biobrick recovery from the iGEM page [2]

1. Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick™-standard part that you want
2. Add 15 uL of diH2O (deionized water)
3. Take 1uL DNA and transform into your desired competent cells, plate out on a plate with the correct antibiotic* and grow overnight. Your goal here is to obtain single colonies.

I intend to use the following protocol for transforming the biobricks into competent cells.

1. Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
2. Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
3. Incubate on ice for 30 minutes
   • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
4. Incubate cells for 30 seconds at 42°C.
5. Incubate cells on ice for 2 min.
6. Add 4 volumes of room temperature SOC (not critical)
7. Incubate for 1 hour at 37°C on shaker.
   • Note: Can also save some time here by reducing incubation to ~45 min.
   • Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
9. Grow overnight at 37°C.

Before the procedure I would need to prepare solid plates with Ampicillin (The vector of the biobrick has a region that confers the bacteria qith Kanamycin resistance). It seems that the proper concentration of Ampicillin is 50 μg/mL.[3]