Team:Leiden/InterLab

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Interlab

InterLab is an iGEM initiative aiming to reduce lab-to-lab variability. This years goal is to normalise fluorescence data to absolute cell counts or colony-forming units (CFUs) instead of OD. As one of many iGEM teams we participated in this study and measured six different GFP expressing E. coli strains, along with a positive and negative control.

Calibration

In order to normalise the fluorescence of GFP in cell measurements, three calibrations were performed. All experiments, including the calibrations, were performed in four replicates. First of all, an OD600 reference was determined to convert the measured absorbance at 600 nm (Abs600) into a comparable OD600 value, such as generally obtained from a spectrophotometer. To that end the Abs600 of both water and a LUDOX CL-X solution (45% colloidal silica suspension) was measured. From these measurements, a conversion factor of 3.679 was obtained between the Abs600 and OD600. The next step is a calibration to convert the obtained absorbance at 600 nm to an estimated number of cells. By measuring samples with different concentrations of monodisperse silica microspheres, a calibration curve was made (Figure 1). As the microspheres have similar size and optical characteristics as cells, this calibration can be used to estimate the cell count in samples.

Figure 1 Calibration curve used for the conversion of absorbance to cell counts. The absorbance was measured in four replicates from monodisperse silica microsphere solution, which has similar size and absorbance properties as cells.

Next, the fluorescence measurement of cells expressing GFP was normalised to the fluorescence of Fluorescein, a fluorescent molecule with a similar excitation and emission profile. A calibration curve for the fluorescence of Fluorescein was made (Figure 2) and used to normalise the fluorescence measurements from cells expressing GFP.

Figure 2 Calibration curve used for the normalisation of fluorescence to that of Fluorescein. The fluorescence of Fluorescein was measured at different concentrations in four replicates.

Cell measurements

E. coli K-12 DH5-α was transformed with six different plasmids (called devices from now on), along with a positive and negative control. The devices have a pSB1C3 backbone and contain promoters, with different expression levels, coupled to a GFP gene (BBa_E0040). More information about the devices and controls can be found on their respective pages in the iGEM BioBrick Registry:

Transformants were kindly provided by the iGEM team of Delft. The Abs600 was measured from an overnight culture of these transformants, which were subsequently diluted to an Abs600 of 0.02. The Abs600 and fluorescence (excitation 485 nm, emission 525 nm, 20 nm bandwidth, gain = 58) were measured from samples at 0 and 6 hours after inoculation. Measurements were performed in 96 well plates in a plate reader (TECAN Spark 10M).

CFU count

In order to determine the amount of colony forming units (CFUs) per 0.1 OD600, overnight cultures of the positive (BBa_I20270) and negative control (BBa_R0040) were diluted to this OD and plated at different dilutions. A total of six dilutions to an OD of 0.1 were made per strain. After 20 hours plates with less than 300 colonies were counted and used to determine the final CFUs per OD600 of 0.1.

Conclusions

Using the calibration curves and the fluorescence and absorbance measurement, the average Molecules of Equivalent Fluorescein (MEFL) per particle was calculated for each of the devices and controls (Figure 3).

Figure 3 Expression of GFP in different E. coli constructs normalised to Molecules of Equivalent Fluorescein (MEFL) per particle. Two colonies of each respective transformations were tested with four independent replicates.

Device 1 showed the highest normalised fluorescence per particle, followed by device 4, 2 and the positive control. Lowest normalised fluorescence was seen in device 6, 3 and the negative control. Device 5 showed a remarkable difference between the two replicates (Figure 3). This was due to both a difference in absorbance and fluorescence measurement, amplified in the difference in MEFL/particle. The reason why both absorbance and fluorescence differed between replicates is unknown.

The average CFUs were calculated using the dilution factor and the amount of colonies counted on the respective plates. Using these results, the average CFUs per OD600 of 0.1 were found to be 3.5 × 107 ± 2 × 10 7 and 3.8 × 107 ± 2 × 107 for the negative and positive control respectively. As the average CFU count is very similar for both positive and negative controls, expression of GFP is not likely to impact viability of cells.

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