iGEM, in its fifth interlab study has focused majorly on reliability and reproducibility of the results. Fluorescein, a reporter, is targeted for the studies performed this year as the intensity observed directly correlates with the number of cells that are producing it. However, the comparison among the data provided by different labs is difficult due to two major reasons: Different units of reporting the data and different ways of processing the raw data so as to suit the labs.
However, iGEM measurement committee this year has taken great steps to turn this into reality.
Initially, there were three basic calibration steps that needed to be followed.
Reference point protocol
- This protocol is used in conversion of the Abs600 values to OD600 values
- This is necessary because the Abs readings are volume-dependent. The fluid in the well defines the path length of the light passing through it.
- The cuvette thickness defines the pathlength in standard spectrophotometer. Ludox is used because this reagent scatters light the least.
- Added 100 microliters of Ludox in wells labelled 1
- Added 100 microliters of dH2O in wells labelled 2
- Measured the readings
- Reported the values
Particle standard curve
- The microspheres obtained display similar properties as that of the cell.
- These are used to convert the Abs600 readings to number of cells.
- Obtain the microspherea from the kit and vigorously shake the solution
- Pipette 96 microliters of this solution to 904 microliters of water
- Prepare serial dilutions of the solution
- Measure the reading at Abs600. Report the readings.
Fluorescein standard curve
- This is used to measure and obtain a standard curve which is further used to convert the cell-based readings to equivalent fluorescein readings (concentration).
- Prepare the stock solution of fluorescein
- Prepare serial dilutions as in the particle standard curve protocol
- Observe and record the data
- Measure the fluorescence
Cell measurement protocol
Colony Forming Units
- This is used to calibrate OD600 to colony forming unit (CFU) counts.
- This protocol assumes that one bacterium gives rise to one colony.
- Grow overnight cultures (16 hours) of the E. coli DH5 α. Dilute the OD to 0.1
- The serial dilutions were prepared as per the protocol and then plated
- The cultures were incubated overnight (16 hours) and the number of colonies were counted
- Assuming that one cell gives rise to one colony, the readings were noted and recorded
- As per the data observed the test device 4 and 5 give the highest values for raw OD data.
- These also give the highest values for the fluorescence readings.
- The MEFL/particle readings for the colony 4 and 5 are also the highest for both 0th hour and 6th hour.
- This means that the colony 4 and 5 have grown very well compared to others.
- The raw data depicts that the colony 4, 5 and 6 have already reached their death phase and hence at the 6th hour reading they display lower values.
- The fluorescence data for the colonies 4 and 5 are highest for 0th hour and for the 6th hour.
- The other colonies might not have grown properly in the given time due to:ul
- Long lag phase (They might have required longer time to adapt to the environment provided)
- Toxicity created by themselves in the environment.