21 August 2008

Dry Lab

• Generated a synthetic video of a bacterium swimming as it would be seen from the microscope, i.e with a noisy background, with realistic cell and background intensities
• Tested the synthetic video with the SpotTracker pluggin for ImageJ: results were unconvincing
• Continued work on modelling the growth curve of bacteria using ODEs

Wet Lab

Cloning

• Double digest (EcoRI and SpeI) of all registry sequences performed and insert sizes checked on gel

Lanes : M = Marker, 1 = Terminator Undigested, 2 = Terminator Digested, 3 = mRFP - Terminator Undigested, 4 = mRFP - Terminator Digested, 5 = AK3 Undigested, 6 = AK3 Digested, 7 = GFP-mut3b Undigested, 8 = GFP-mut3b Digested, 9 = CAT Undigested, 10 = CAT digested

No insert can be observed for the terminator, this insert is too small to be visualised but a short run on a gel will be used to check for the presence of this insert tomorrow. The mRFP - terminator insert is approximately 1kb and can be seen, similarily GFP - Terminator is approximately 1kb (also shown). AK3 does not appear to have an insert (which would be correct as the nromal insert is lethal to E.coli and there is a 600bp insert clearly visible from the CAT digest. All registry sequences appear to be the correct size, indicating that they are probably what they are meant to be.

B.subtilis

• The B.subtilis was successfully transformed using transformation of protocol 2, [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation_protocol_4#Transformation_Protocol_2| Click link here for protocol]. However, colonies were seen on the plates were no DNA was used for transformation. The most likely cayse of this is contamination whilst carrying out the electroporation. To check that the LB agar plates contained streptinomycin we picked a transformed colony and streaked it out on a freshly made LB agar plate containing streptinmycin.
• In addition the transformation protocol 1 was carried out, [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/Transformation_protocol_2| click here for a link to the protocol]. The principle of this protocol is that a culture is grown to a high O.D.₆₀₀, this induces the stress pathways in B.subtilis and induces natural competence. These high cultures are diluted and then incubated with suitable concentrations of DNA. Transformed B.subtilis can then be plated out and grown on antibiotic resistant plates. Today B.subtilis were grown to an O.D.₆₀₀ of 2.0 and incubated with 40ng to 400ng of pDR110 DNA.