Team:NTNU Trondheim/InterLab

One of the biggest problems in synthetic biology is that there is a big variability in the results you get from different labs. The goal of the interlab study is to minimize this variability, so that the measurements are both reliable and repeatable for labs all over the world. This year’s interlab study focus on the question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? To answer this question two approaches were used [1]:

  1. Converting between absorbance of cells to absorbance of a known concentration of beads.
  2. Counting colony forming units from the sample.

Calibration

There were made three sets of calibration measurements: OD600 reference point, particle standard curve and fluorescein standard curve.

The first calibration measurement was to get a conversion factor so that we could easily convert absorbance (Abs600) to OD600. Here we used the LUDOX CL-X. The results were:

Table 1: Abs600 using LUDOX CL-X and H2O for 4 replicates. The corrected Abs600 is the difference between Arith. means for LUDOX CL-X and H2O, and together with a reference OD600 was used to calculate the conversion factor OD600/Abs600.
LUDOX CL-X H2O
Replicate 1 0.057 0.038
Replicate 2 0.058 0.038
Replicate 3 0.056 0.038
Replicate 4 0.056 0.038
Arith. Mean 0.057 0.038
Corrected Abs600 0.019
Reference OD600 0.063
OD600/Abs600 3.338

For the particle standard curve, a dilutions series of monodisperse silica microspheres was prepared and the absorbance was measured in a Tecan Infinite M200 Pro plate reader. This curve was used to convert absorbance to number of cells. Figure 1 shows the linear particle standard curve, and a mathematical relationship between absorbance and number of particles was estimated:
y = 3*10-9x + 0.0401
with a value of R² = 0.9995. A particle standard curve in log scale was also plotted as shown in Figure 2.

Figure 1: Particle standard curve. The mathematical equation of the standard curve was estimated to be y= 3*10-9x + 0.0401, with R² = 0.9995.
Figure 2: Particle standard curve in logarithmic scale

A dilution series for the fluorescein standard curve was also made. The fluorescence was measured in a Tecan Infinite M200 Pro plate reader with an excitation wavelength 494nm and an emission wavelength 525nm. Figure 3 shows the relationship between fluorescence and the concentration of fluorescein. A plot in logarithmic scale was also made (Figure 4).

Figure 3: Fluorescein standard curve. The fluorescence was measured with an excitation wavelength 494nm and an emission wavelength 525nm.
Figure 4: Fluorescein standard curve in logarithmic scale

Cell measurements

In this study, we transformed Escherichia coli DH5α with the following plasmids from the iGEM distribution kit:

Table 2: An overview of used Biobrick parts and their location in the iGEM distribution kit for the Interlab study.
Device Part number Plate Location
Negative control BBa_R0040 Kit Plate 7 Well 2D
Positive control BBa_I20270 Kit Plate 7 Well 2B
Test Device 1 BBa_J364000 Kit Plate 7 Well 2F
Test Device 2 BBa_J364001 Kit Plate 7 Well 2H
Test Device 3 BBa_J364002 Kit Plate 7 Well 2J
Test Device 4 BBa_J364007 Kit Plate 7 Well 2L
Test Device 5 BBa_J364008 Kit Plate 7 Well 2N
Test Device 6 BBa_J364009 Kit Plate 7 Well 2P

The day after, two colonies from each plate were inoculated in LB-medium + Chloramphenicol. The cells grew overnight. OD600 was measured and the cultures were diluted to a target OD600 of 0.02 in a final volume of 12 mL. Abs600 and fluorescence were measured after 0h and 6h incubation with four replicates.

Net Abs600

Figure 5:Net Abs600 at 0 h and after 6 h incubation of cell cultures that originated from colony 1. The cell cultures either carried Device 1, Device 2, Device 3, Device 4, Device 5, Device 6, positive control or negative control.
Figure 6: Net Abs600 at 0 h and after 6 h incubation of cell cultures that originated from colony 2. The cell cultures either carried Device 1, Device 2, Device 3, Device 4, Device 5, Device 6, positive control or negative control.

Figure 5 and Figure 6 show the Abs600 measured at 0 h and after 6 h of incubation from cell colony 1 and cell colony 2, respectively. By comparing the absorbance between the two colonies after 6 h incubation, the cells growth with different rate. However, for both cell colonies, the lowest net absorbance was detected from cells with device 1 or device 5 compared with the cells that did not carry either of these two devices.

Net Fluorescein a.u

Figure 7:Net Fluorescein a.u at 0 h and after 6 h incubation of cell cultures originated from colony 1. The cell either carried device 1, device 2, device 3, device 4, device 5, device 6, negative control or positive control.
Figure 8:Net Fluorescein a.u at 0 h and after 6 h incubation of cell cultures originated from colony 2. The cell either carried device 1, device 2, device 3, device 4, device 5, device 6, negative control or positive control.

Figure 7 and Figure 8 show the fluorescence of the different devices at 0h and after 6h incubation for colony 1 and colony 2, respectively. The highest net fluorescein a.u was detected from cells which carried device 4, while cells with device 3 had the lowest net fluorescein a.u (Figure 7 and Figure 8). However, the net fluorescein a.u measured from colony 1 tend to be higher, compared with colony 2.

µM Fluorescein/OD

Figure 9:µM Fluorescein/OD at 0 h and after 6 h incubation of cell cultures originated from colony 1. The cell either carried device 1, device 2, device 3, device 4, device 5, device 6, negative control or positive control.
Figure 10:µM Fluorescein/OD 0 h and after 6 h incubation of cell cultures originated from colony 2. The cell either carried device 1, device 2, device 3, device 4, device 5, device 6, negative control or positive control.

The highest µM Fluorescein/OD was detected from cells which carried device 1, 4 or 5. On the other hand, cells which carried device 3 had the lowest µM Fluorescein/OD, which is comparable with the negative control. Figure 9 and Figure 10 show the µM Fluorescein/OD at 0 h and after 6 h incubation of cell cultures from colony 1 and colony 2, respectively.

Molecules of equivalent fluorescence level per particle (MEFL/ particle)

Figure 11:Molecules of equivalent fluorescence level per particle (MEFL/particle) at 0 h and after 6 h incubation of cell cultures originated from colony 1. The cell either carried device 1, device 2, device 3, device 4, device 5, device 6, negative control or positive control.
Figure 12:Molecules of equivalent fluorescence level per particle (MEFL/particle) at 0 h and after 6 h incubation of cell cultures originated from colony 1. The cell either carried device 1, device 2, device 3, device 4, device 5, device 6, negative control or positive control.

Like the µM Fluorescein/OD, device 1, 4 and 5 had the highest MEFL/ particle value, while device 3 had the lowest MEFL/ particle value (Figure 11 and Figure 12).

Colony forming units

The second approach of this interlab study was to find colony forming units (CFUs) per 0.1 OD600 E. Coli cultures. Starting samples were prepared from the positive and negative control by diluting the overnight culture 20 times until OD=0.1 for the cell culture minus the blank measurement. Dilution series was prepared and 36 plates were aseptically spread with dilution series 3, 4 and 5.

Given that the cultures were diluted to target OD600 = 0,1. The Abs600 = 0,0678 (including blank) was obtained by using the factor OD600/ Abs600 = 3,338 to convert OD600 to Abs600. According to the standard particle curve, the predicted number of cells will be 0,93*107. By comparing the values of CFU/mL from Table 5 with the predicted number of cells (0,93*107), these values have a relatively large variance, and a large deviance from the predicted number of cells.

Table 3: Dilution factors for dilution series 3, 4 and 5.
Dilution 3 Dilution 4 Dilution 5
Final dillution factor 8*104 8*105 8*106

The plates were incubated at 37°C overnight. The day after, the colonies were counted and the results are given in table 4 and 5:

Table 4: The average values of counted colonies from two cultures of positive and negative controls with different final dilution factor.
Positive control Negative control
Culture 1, dilution 3 194.3 117.3
Culture 1, dilution 4 7.7 31.0
Culture 1, dilution 5 1.3 2.3
Culture 2, dilution 3 104.0 87.0
Culture 2, dilution 4 18.7 5.7
Culture 2, dilution 5 1.3 0.7
Table 5: Colony forming units (CFU) per mL of an OD600 = 0.1 starting samples of two cultures of positive and negative controls with different final dilution factors.
Positive control [CFU/mL] Negative control [CFU/mL]
Culture 1, dilution 3 1.6*107 0.9*107
Culture 1, dilution 4 0.6*107 2.5*107
Culture 1, dilution 5 1.1*107 1.9*107
Culture 2, dilution 3 0.8*107 0.7*107
Culture 2, dilution 4 1.5*107 0.5*107
Culture 2, dilution 5 1.1*107 0.5*107

Conclusion

The results obtained from this experiment, indicate that the two approaches for reducing the lab-to-lab variability in fluorescence measurement, do not seem to be reliable. Neither method 1 nor method 2 gave results that could return to the cell concentration in the sample, due to a relatively large variance and deviation.

Sources: [1] http://2018.igem.org/Measurement/InterLab