Imperial College London/Notebook/23 September 2009

Nuri: - Plotted curves GFP/normalized OD - Observations: Control is dodgy... negative values came up, so need to re-do in wetlab! - Diauxic growth model: Writing up and reviewing model and simulations

Testing team

Harvard GFP (1st run)

• Conclusions:
  • Raw fluorescence seems to increase with time at 37°C while, at 28°C, it seems to stay stable

• Problems:
  • Did not run enough data points to see the fluorescence of the shifted sample reach a steady state
  • Cannot really conclude that the increase of fluorescence is due to an increase of expression as opposed to simple cell growth. This can be seen by the similarity of data on the normalised graph (bottom right, figure 1).

OD calibration 2nd run 23rd Sep

Protocol considerations

• Cells were grown overnight in 10ml of M9 media
• The overnight culture had an OD of around 1.5. This was taken as the uppermost range of OD, and diluted down until 0.4
• subsequently diluted to 10^-7 and 10^-8 for higher OD, and 10^-6 and 10^-7 for lower OD values
• The overnight culture and all dilutions were kept on ice
• 50uL of the culture was inoculated onto each plate
• Duplicate of measurement was done

Results

Download raw data

Conclusions

• The OD seems to correlated with the colony count, but the variation in the colony counting is huge

• Doing on ice stablises the colony number, so plates do not become confluent.
• 10^-7 and 10^-8 produced too few colonies, so lower dilutions should be attempted

Suggestions/Improvements

• Suggestions:
  • Inoculate 100uL
  • Lower dilution factors (10^-4 to 10^-6)