14 August 2008

Wet Lab

Cloning

• Parts taken from the registry and transformed by electroporation into XL1-Blue E.coli along with XL1-Blue controls and grown on Kanamycin and Ampicillin plates. Parts taken:
  • C0012 (LacI)
  • J31005 (Chloraphemicol acetyltransferase)
  • B0015 (Double terminator)
  • J04630 (GFP and Double Terminator)
  • I13401 (mRFP and Double Terminator)

B.subtilis

• The transformation protocol 2 was carried out today, [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation_protocol_4#Transformation_Protocol_2| Click link here for protocol]
• The basic principle of this protocol is that competent cells are prepared by growing a culture to a high O.D.₆₀₀ and then performing electroporation on these competent cells.
• Today we grew the competent cells, we used an O.D.₆₀₀ of 1.5 before we harvested them. In addition electroporation was carried out using the plasmid pDR110. Previously we had mini-preped this to give a stock of 40ng/ul. We transformed with 40ng, 120ng, 200ng and 400ng in a volume of 10ul (water was used to make up to 10ul).
• Transformed cells were plated out and placed into the 30^oC incubator.

Dry Lab

• Completed A More Complex Example of Bayesian Parameter Estimation of Tutorial 2.
• Sourced for better tracking algorithm, found SpotTracker which is more accurate than ParticleTracker.
• Went for microscope training, obtained 2 x videos on B.Subtilis motility, stored on server with James.
• Discussion with Dr. Suhail ( from Structural Bioinformatics Group,Imperial College London) on the computing requirements to analyse the cells motility. We will be allocated a linux 64-bit workstation, that we will be able to access remotely via SSH.

Microscope

• Chris, Clinton, James and Prudence received microscope training on Widefield 1, recording two 60s videos of B.subtilis swimming for analysis
• Determined that best results would be obtained by bringing a B.subtilis overnight culture to the microscope facility and diluting 100 fold.