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

-Designed two plasmids:

-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

Class Registry Part Kit Location Tube Label
Promoter BBa_J23100 2008 1002-10E A
RBS BBa_B0030 2008 1002-5G B
Stop BBa_B1002 2008 1016-8A C
Stop BBa_B1006 2008 1016-8D D
Control pUC19 E
  • 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 )