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

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

Wet Lab Work

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 )