Team:Madrid-OLM/InterLab

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The InterLab Study

Results

Introdution

iGEM’s Interlab is the world biggest synthetic biology interlaboratory study. Replicability is key in science due to the importance of the comparison of data between researchers and their experiments internationally. This is currently a major obstacle during the transition between theory and experimentation. The considerable asset of the Interlab is the reduction of variability, which can ease the experimental reproduction of a synthetic biology protocol. That is why since 2014, teams worldwide have contributed making a baseline protocol for synthetic biology fluorescence measurements.

Objetive

In iGEM’s fifth International Interlaboratory Study the purpose, as in previous ones is reducing variability in synthetic biology measurement. The main focus of this interlab study is to develop a direct method to evaluate GFP fluorescence in a bulk of cells. This should be more precise than the actual method, which divides the whole fluorescence value by the number of cells in a sample. The use of same-sized E.coli beads for calibration can let us convert absorbance measurements into concentration ones. After, we will make a normalization of the cell count of colony-forming units that would be a way of increasing the measurement accuracy. The synergy of the CFUs and the beads would facilitate the success of the new method of GFP fluorescence measurement.

Plate Reader

• Brand and model: ThermoScientific Varoskan Lux.

• Absorbance and fluorescence: The plate reader is capable of measures both.

• Pathlenght correction: Pathlenght correction: yes, but it will be disabled.

• Temperature settings: It has temperature settings and the temperature can be set from 4°C to 45°C.

• Filters from the instrument: It has been used an excitation of 485nm and a 520 emission.

• Top or botton optics: It has both.

Protocol

For each section we have use the Plate Reader Protocol.

Results

Calibrations

• Calibration 1

  The aim of the first calibration is to obtain a conversion factor. This conversion factor is going to give the cell volume number according to the absorbance. The main advantage of measure optical density against absorbance is that, the first one is independent from volume, but the latter is dependant.

  Materials

  • -1mL LUDOX CL-X (provided in kit).

  • -ddH2O (provided by team).

  • -96 well plate, black with clear flat bottom preferred (provided by team).

  Results

  

  Table 1. It is shown the OD600 measurement by the first calibration experiment. The corrected Abs600 is made subtracting H2O measurements from LUDOX CL-X ones. The OD600/Abs600 result (3,652) is the multiplication factor, which you have to use after a cell density measurement with the plate reader to have a correct analysis.

• Calibration 2

  One of the objectives of Interlab is to be able to know the number of cells from the absorbance. In second calibration it is made a Silica beads standard curve. This microspheres have a similar volume and optical density than cells, so the measurements can give a precise data about the number of cells.

  Materials

  • -300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10^8 microspheres).

  • -ddH20 (provided by team).

  • -96 well plate, black with clear flat bottom preferred (provided by team).

  Results

  

  Table 2. The results correlate with the serial dilutions that have been made. The number of particles is reduced until there are practically no particles in columns 10 and 11. Such results are similar to those in column 12 that only have ddH2O.

  

  Figure 1. The particle Standard Curve is very linear and has superfluous error bars.

  

  Figure 2. The Particle Standard Curve (log scale) has a very precise logarithmic curve.

  

  Table 3. Table with Particles/OD factor.

• Calibration 3

  Plate readers are a very accurate measurement machines. However, one of the main disadvantages they have is the difficulty when comparing the data with other plate readers. In the third calibration, are using the fluorescein protein to do a fluorescence standard calibration curve. The results will make possible to convert cell measurements in fluorescent concentration. Also this will be independent of the model of plate reader used.

  Materials

  • -Fluorescein (provided in kit).

  • -10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team).

  • -6 96 well plate, black with clear flat bottom (provided by team)

  Results

  

  Table 4. Serial dilution fluorescence results.

  

  Figure 3. Fluorescein Standard Curve.

  

  Figure 4. Fluorescein logarithmic standard curve.

  

  Table 5. Molecules of equivalent fluorescen/ a.u.

Cell Measurement

After the three calibrations, the cells were measured. The E.coli strain K12 DH5-alpha was used.

Table 6. 0 and 6 hour fluorescence raw readings.

Table 7. 0 and 6 hour Abs600 raw readings.

Table 8. Net Abs600 measurements.

Table 9. Fluorescence per OD.

Table 10. Unit Scaling Factors in fluorescence per OD.

Colony Forming Units

The CFU protocol was made assuming that each bacterial cell will form one colony. Knowing this it was done the colony counting and the calculations for the CFU.

Table 11. Colony counting and CFU.

Table 11. Colony counting and CFU.