Imperial College London/Notebook/2 October 2009

Testing team update

Harvard-GFP

• There is a difference in fluorescence between 28°C and 37°C after compensating for OD.
• Need to adjust the gain so that fluorescence doesn't saturate
• 23rd September
• 30th September

OD calibration

• Preliminary results show that cell number varies with OD. However, the right dilution and enough replicates to overcome the huge variation are required.
• 17th of September (up)
• 23rd of September (awaiting results)
• 25th of September
• 2nd October (awaiting results)

Secondary carbon sources

• There appears to be a plateau in cell number after 6 hours with 0.05% Glucose. However, more data is required to determine if this is the switch point.
• 8th September (up)
• 10th September (up)
• 1st October

GFP pH

• 18th September (awaiting data analysis)

IPTG growth

• IPTG has no effect on cell growth under the concentrations tested (100uM to 5mM)
  • This is despit the dual effects of possible toxicity and protein production stress by IPTG on Lac-RFP constructs

• 16th September (up)

Testing

OD calibration 4th run 2nd Oct

Protocol considerations

• Cells were grown overnight in 10ml of M9 media
• The cells were allowed to grow until a high OD in the late morning, then placed in cold room
• OD of around 0.4 to 1.2 was made up, and subsequently diluted up to 10^-4 to 10^-6
• 100ul of cells were inoculated into each plate
• The OD of the overnight cells were measured again after 3 hours on ice to check if cell number changes during dilution process

Results

Conclusions

• After 3 hours on ice, the OD stays relatively constant
  • Shows that number of cells during OD measurement and during plating stay the same. Therefore, OD correlates with the number of cells that are plated.

Suggestions/Improvements

Waiting to be uploaded