Team:Macquarie Australia/InterLab
Background
Synthetic biology is a type of engineering and has also been called engineering biology. As with all engineering disciplines, synthetic biology requires reliable and repeatable measurements. For the past few years, the international interlaboratory study has been aiming to minimise the variability between fluorescence measurements from laboratories around the world, using the green fluorescent protein gene (GFP). By now, the studies have established a baseline for the replicability of these measurements, as well as identifying likely sources of error between laboratories.
Aim for 2018
This year, the ChIVes have volunteered to participate in the InterLab study aiming to set the baseline for comparing fluorescent data between laboratories. There are at least two limiting factors that make this comparison difficult;
- 1. Interpretation of fluorescence results in different units between laboratories
- 2. Variability in the bulk measurements of a population of cells, due to unknown number of the cells in the testing means (such as a plate reader)
Hence the question to be assessed arises; Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?
The data were obtained using the protocol provided by the iGEM Measurement Committee which will be used to make comparisons between all the participating teams, in order to address the hypothesis.
Materials and Methods
Click Here for the full protocol
Calibrations
1. OD600 Reference point - LUDOX protocol
The 600 nm absorbances of 100% LUDOX and H2O replicates in a well plate were measured and recorded to calculate the OD600 reference point.
2. Particle Standard curve - Microsphere protocol
Serial dilutions of monodisperse silica microspheres were prepared and were used to measure the absorbance (Abs600) using the BMG Labtech Fluostar Optima plate reader and to construct particle standard curves of particle concentration (linear and log scale). Since the microspheres are of known concentration and can be used to mimic Escherichia coli DH5α cells, the standard curves can be used to convert Abs600 measurements to an estimated number of cells.
3. Fluorescence standard curve - Fluorescein Protocol
Serial dilutions of fluorescein were prepared in four replicates, which was used to measure the fluorescence using the BMG Labtech Fluostar Optima plate reader (excitation at 485 nm, emission at 520 nm). The measurements can be used to construct a standard curve of fluorescence for fluorescein concentration, which can be used to convert E. coli DH5α cell based readings to an equivalent fluorescein concentration.
Cell Measurement
K-12 E. coli DH5α cells were transformed with the plasmids supplied (all in pSB1C3 backbone) in the 2018 iGEM Kit Plate 7, which contain different promoters before the fluorescence gene.
| 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 |
- 1. The transformed cells were grown on chloramphenicol supplemented agar plates and two colonies from each device were used to prepare liquid cultures which were allowed to grow overnight.
- 2. The liquid culture samples were diluted to Abs600 = 0.02
- 3. Absorbance and fluorescence measurements were obtained fusing the BMG Labtech Fluostar Optima plate reader
- 4. The remaining samples were incubated for 6 hours at 37°C and 220 rpm.
- 5. Absorbance and fluorescence measurements were obtained fusing the BMG Labtech Fluostar Optima plate reader following the 6 hour incubation.
Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures
The CFU protocol was used to count colonies for the two “positive control” (BBa_I20270) liquid cultures and and two “negative control” (BBa_R0040) liquid cultures. The same overnight cultures were diluted to OD600 = 0.1 and were then used for further serial dilutions. The diluted samples were plated in LB agar +Cam plates and the CFU/mL/OD was calculated.
The absolute cell count was obtained by converting between the absorbance of E. coli DH5α cells to absorbance of a known concentration of beads, while the CFUs were calculated based on the number of colonies present in LB + chloramphenicol media of the positive and negative control samples.
Results
Calibrations
1. OD600 Reference point - LUDOX protocol:
The reference point was determined to be OD600/Abs600 = 1.527
2. Particle Standard curve - Microsphere protocol
The following standard curves were obtained from the calibrations and were used for determination of the absolute cell count of E. coli DH5α cells in the samples, based on Abs600 (Figure 1, Figure 2)
Figure 1. Particle standard curve generated by measuring the absorbance (Abs600) of serial dilutions of monodisperse silica microspheres using the BMG Labtech Fluostar Optima plate reader.
Figure 2. Particle standard curve (log scale) generated by measuring the absorbance (Abs600) of serial dilutions of monodisperse silica microspheres using the BMG Labtech Fluostar Optima plate reader.
3. Fluorescence standard curve - Fluorescein Protocol
The following standard curves were obtained from the calibrations and were used for determination of the fluorescein concentration in the transformed E. coli DH5α cells, based on fluorescence measurements (Figure 3, Figure 4).
Figure 3. Fluorescein standard curve generated by measuring the fluorescence (au) of serial dilutions of the fluorescein sample provided, using the BMG Labtech Fluostar Optima plate reader.
Figure 4. Fluorescein standard curve (log scale) generated by measuring the fluorescence (au) of serial dilutions of the fluorescein sample provided, using the BMG Labtech Fluostar Optima plate reader.
Cell measurement
The raw results from the plate reader were recorded for absorbance and fluorescence at 0 and 6 hours and were used to obtain the following graphs:
Figure 5. Raw results generated by measuring the absorbance (Abs600) of serial dilutions of E. coli DH5α cells transformed with the different testing devices provided. The absorbance was measured using the BMG Labtech Fluostar Optima plate reader. The cells were grown overnight and were diluted to Abs600 = 0.02, which is the absorbance at 0 hours. Then, they were further incubated for 6 hours at 37°C and 220 rpm, and the absorbance was measured again.
The absorbance of the blank got higher from 0 to 6 hours (Figure 5), which probably indicates contamination of the sample. This caused the absorbances of Device 1, Device 4 and Device 5 to be negative after subtracting the blank measurement. Therefore, for standardisation of the results, we only used the Negative control, the Positive control, Device 2, Device 3 and Device 6.
Figure 6. Raw results generated by measuring the fluorescence (au) of serial dilutions of E. coli DH5α cells transformed with the different testing devices provided. The fluorescence was measured using the BMG Labtech Fluostar Optima plate reader. The cells were grown overnight and were diluted to Abs600 = 0.02, which shows the fluorescence at 0 hours. Then, they were further incubated for 6 hours at 37°C and 220 rpm, and the fluorescence was measured again.
The measurements of fluorescence (Figure 6) were as expected with the blank and the negative control remaining stable at 0 and 6 hours and the positive control showing increased growth after the 6 hour incubation. The different devices showed different fluorescence, however based on the results of Abs600, the only devices that were used for standardisation of the results, were the negative control, the positive control, Device 2, Device 3 and Device 6.
By fitting the raw results in the standard curves of the nano-particles and the fluorescein we obtained the graphs of μΜ Fluorescein/OD at t=6 hours (Figure 7) and MEFL/particle at t=6 hours (Figure 8).This was done by subtracting the blank measurements from the 0 hour measurements and then the 0 hour measurements from the 6 hour ones.
Figure 7. The raw results obtained from the fluorescence of the different devices were fit into the fluorescein standard curve, using the OD600/Abs600 as a reference point. To obtain this graph, the blank measurements were subtracted from the measurements of 0 hours. Then, the 0 hours measurements were subtracted from the 6 hours to determine the difference in GFP (fluorescence) concentration. Only the devices with higher absorbance than the blank were plotted.
Figure 8. Results of MELF/particle at 6 hours were obtained by standardising the raw results of absorbance and fluorescence, using the unit scaling factors of “Particles / Abs600” and “MEFL / a.u.”. The blank measurements were subtracted from the measurements of the devices at 0 hours, which in turn were subtracted from the measurements of 6 hours for determination of difference in absorbance and fluorescence. Only the devices with higher absorbance than the blank were plotted.
Colony Forming Units per 0.1 OD600 E. coli cultures
For the dilution with the dilution factor of 8 x 104 the colonies were too many to count (TMTC) and hence it is expected that they were at least 500 colonies in each plate. The dilution with dilution factor of 8 x 105 was the first that allowed for colony count in the LB media + Cam plates which was decreased following another 10-fold dilution (dilution factor of 8 x 106 ), as seen below (Figure 9).
Figure 9. Colony forming units present in LB + cam media. The negative (colony 1A, 1B) and positive (colony 2A, 2B) control devices were grown overnight and then diluted to OD = 0.1. Serial dilutions were performed in triplicates for each colony and the dilutions of 8x10-3 ,8x10-4 and 8x10-5 were plated into LB + cam media (100 ul). Following incubation at 37°C overnight the colony forming units were calculated. For the dilution 8x10-3 the colonies formed were too many to count (TMTC) and the count decreased after the further 10-fold dilutions.
Conclusions
Volunteering for the interlab was a fun and valuable experience to be part of. We suggest that the results could be presented by subtracting the blank measurement from its corresponding replicate at 0 hours and then the 0 hours from the respective measurement at 6 hours, to show the difference in absorbance and fluorescence at 6 hours.
It is expected that the results presented by our team are consistent with other teams’ results and combined, they can be used to determine whether the lab-to-lab variability in fluorescence measurements can be reduced, by normalising to absolute cell count or CFU, instead on OD.
