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From 2010.igem.org

Contents 1 Lab work 2 Emulsifier 3 Meeting Minutes 3.1 Labwork 3.2 Others

Lab work

The deadline is now really closing in. Everyone is super busy in the lab to reproduce as much data as possible to make our results ever prettier. So, less time is spend on blogging :)

Emulsifier

We tried to isolate the emulsifier proteins from the pellets we made last friday. It was a bit disappointing, but expected, that the total protein yield was lower than last time. This is probably caused by the fact that we stopped the growth of the culture before it reached the stationairy phase at OD600 of 0.6. The total finale volume per sample was 1 mL.

The rough protein yields were determined by Bradford analysis:

#	Description	[Protein](mg/ml)
1	AlnA 37 C	1.55
2	AlnA 30 C	2.29
3	Control 37 C	1.78
4	Control 30 C	2.42

So it looks like Esengul was right about the higher IPTG induction at lower growth temperatures.

The samples were used in the emulsifier assay, to determine their emulsifying capacity. I used 0.75 mg of protein in each sample. This is the first draft of the data analysis:

Meeting Minutes

Labwork

Hugo:

• ADH – significant difference between our strain and empty plasmid – Dodecanol and NAD, measure production of NADH. Standard unit: Micromol/min/mg protein (value – would be enough to support (some) growth)
• pCaif – do it again… results don’t prove that our BioBrick works. Leaky promoter? Grow on Ethanol (2-5 gr/L) to have the promoter switched on all the time. Also try higher concentrations of Glucose – maybe 2 g/L is already keeping the GFP production on?
• Solvent tolerance: done (not an empty plasmid, but K12…) Emtpy plasmid would be better

Thias:

• LadA – add substrate as last, before you add this this shows a background, after you add the substrate look at the slope – this is called Substrate specific conversion.
  • Inhibition of heptadecane?
  • Has not been sequenced yet…
• AlkB – resting cell assay, GC was negative
  • New GC running now
• RBS characterization
  • Our results are different from Warsaw’s results
  • They use stationary cells, we use growing cells – standard is exponential
  • Do it again – has only been done once

Pieter: Emulsifier Eva: Sending BioBricks, some duplicates

Others

• Omics – Cell net analyzer, put links to the tools, “collaboration”
• TU Delft Wiki lockdown next week Monday
• Safety Photo!
• Everybody should do a quiz prior to iGEM, make the quiz