IGEM:Cambridge/2008/Notebook/Voltage/2008/07/17

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Progress
-Ordered mutant E.coli chassis to test: Kch-, KdpD-, KdpE-, KdpF-E-

-Designed two plasmids:


 * Kdp Plasmid


 * Glu Gated channel Plasmid

-Sourced V.harveyi BB170 culture to obtain glutamate-gated potassium channel gene

-Modelled protein structure

-Designed primers for extraction of Kdp Operon from E.coli and Glu Gated channel gene from V.harveyi

-Meeting with Julia Davies

-Decided on oxygen electrode for small voltage change measurements

-Extracted BioBricks from registry to use in plasmids:


 * Promoter BBa_J23100


 * RBS BBa_B0030


 * Terminators BBa_B1002 and BBa_B1006

Biobrick Extraction

 * Add 5μL of EB into Eppendorf tubes with punched spots
 * Warm punched spots in 50°C for 20 minutes
 * Spin down for 3 minutes at 15,000 g


 * Chilled 2μLmL tubes
 * Add 2μL of DNA + EB solution (1μL of Control)
 * Add 50μL of Cells ( Top 10 - B ; DH5α - A,C,D,E )


 * Ice for 30 minutes
 * Heat shock at 42°C for 60 seconds
 * Ice for 2 minutes
 * Add 600μL of SOC
 * Incubate at 37°C for 2 hours


 * Prepare Agar plates : Add 200μLof Amp (100mg/mL) into 200mL of LA ( Final conc. of 100μg/mL )


 * Plate Neat(1:1) and Diluted (1:10) for each tube A,B,C,D,E ( 1:10 is prepared by adding 10μL from tubes and 90μL SOC )


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