Team:UCopenhagen/InterLab
InterLab
1. Introduction
PharMARSy decided to participate in the fifth InterLab study. The InterLab study, organized by the measurement committee of iGEM, was an option to obtain the bronze medal requirement #4. The goal of this year’s InterLab study was to collect data to help answer 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?”
The purpose of the InterLab study is to promote standardization of synthetic biology by creating a solid “measurement tool” for measurements of GFP to be used by the iGEM community but also researchers in general. To read more about the InterLab in general please click here.
1.1 Protocols and data submission
A protocol was provided by the measurement committee which included information of how to carry out the three calibrations and the two experiments that constituted this year´s InterLab study. An Excel sheet was also provided whereto most of the data were to be collected. Information of the details of the experiments and some extra data was handed over to the measurement committee through online submitted forms.
1.2 Plate reader and plates
To measure the absorbance and fluorescence we used the plate reader SpectraMax M5. The 96-well plates used had black sides and a clear, flat bottom. The machine was set to shake for five seconds before the read. As the machine was not able to keep the temperature fixed at a specific temperature (within the relevant temperature range of these experiments), measurements were taken between 20-30 oC.
For absorbance measurements, absorbance of the samples were measured at 600 nm. Pathlength correction was set to ‘off’. For fluorescence measurements, samples were measured as a top read with an excitation wavelength at 485 nm and an emission wavelength at 525 nm (cutoff at 515 nm).
2. Calibrations
Three different calibration tests were performed to obtain standard curves so that the collected data can be compared with the data from other laboratories. This is possible, as the standard curve is based upon known parameters that is expected to act the same way in all the laboratories.
2.1 Ludox
In this calibration, abs600 of LUDOX CL-X (45% colloidal silica suspension) and water was measured. The LUDOX CL-X solution was provided in the InterLab test kit and was not further processed before the measurement.
This calibration was performed to obtain a conversion factor using LUDOX CL-X as a reference point that can be used to convert abs600 data into a comparable OD600 measurement.
We obtained the following data:
| LUDOX CL-X | H20 | |
|---|---|---|
| Replicate 1 | 0.057 | 0.035 |
| Replicate 2 | 0.061 | 0.035 |
| Replicate 3 | 0.055 | 0.035 |
| Replicate 4 | 0.053 | 0.035 |
Table 1: Absorbance measured for 4 replicates at 600 nm for the LUDOX CL-X solution and H2O.
2.2 Silica Beads
In the second calibration the abs600 was measured for solutions of silica beads and of water. The silica beads were provided in the InterLab test kit and herefrom a microsphere stock solution was made. This solution was then used to make a dilution series.
This calibration was made to create a standard curve of different microspheres concentrations (which are alike Escherichia coli) to an abs600. This makes it possible to convert abs600 to a number of cells.
We obtained the following data:
Table 2: Absorbance measured at 600 nm for samples containing different numbers of silica beads (from 0 to 2.35*108 particles). Measurements were performed with 4 replicates.
Figure 1: Particle standard curve showing mean absorbance from four replicates measured for different concentration of silica beads (table 2). The X-axis shows different concentrations of silica beads and the Y-axis show the absorbance.
Figure 2: Particle standard curve showing log(mean absorbance) from four replicates measured for different concentration of silica beads (table 2). The X-axis shows different concentrations of silica beads and the Y-axis show the absorbance on a log scale.
2.3 Fluorescein
In the third calibration, fluorescence (excitation: 485 nm, emission: 525 nm) was measured for fluorescein and of Phosphate Buffered Saline (PBS) with a pH of 7.45.
The fluorescein solution was provided in the InterLab test kit. First, a fluorescein stock solution was made and this solution was then used to make a dilution series.
This calibration was performed to obtain a standard curve of fluorescence specific for our plate reader, and was done to make it possible to compare the data made in these experiments with data from other iGEM teams, as fluorescence values varies broadly with different instruments.
We obtained the following data:
Table 3: Fluorescence measured for different concentrations of fluorescein (0 µM to 10 µM) at excitation: 485 nm, emission: 525 nm. This was replicated four times (Replicate 1-4). N/A corresponds to saturation of the plate reader.
Figure 3: Fluorescein standard curve showing mean fluorescence of four replicates for fluorescence of different concentrations of fluorescein (0 µM to 10 µM). Excitation: 485 nm, emission: 525 nm. The X-axis shows the different concentrations of fluorescein and the Y-axis shows the fluorescence.
Figure 4: Fluorescein standard curve showing log(mean fluorescence) of four replicates for fluorescence of different concentrations of fluorescein (0 µM to 10 µM). Excitation: 485 nm, emission: 525 nm. The X-axis shows the different concentrations of fluorescein and the Y-axis shows the fluorescence on a log scale.
3. Absorbance and fluorescence of transformed cells
In this experiment, bacteria were transformed with plasmids containing different promoters for GFP. After the transformation two colonies were picked and an overnight culture with each colony was made. The overnight culture was diluted to an abs600 of 0.02 in a total volume of 12 mL. The absorbance and fluorescence were then measured of this solution before (t=0 hours) and after incubation (t=6 hours).
Luria Bertani (LB) media was used throughout the experiment and the antibiotic used as selective marker was chloramphenicol (Chlor).
3.1 Plasmids of transformants
The bacteria used in this experiment was Escherichia coli strain DH5α. Using the iGEM Parts Distribution Kit Plates, eight transformations were made containing the following:
Figure 5: Overview of the different plasmids used for transformation of E. coli strain DH5α. Each plasmid (Device), the reference to the iGEM registry (Part number) and where the plasmid can be found in the distribution kit (Plate + Location) can be seen.
3.2 Absorbance
Absorbance at 600 nm was measured for two colonies of each device. For each of the colonies, four technical replicates were measured. This measurement was performed at the immediate start of the incubation (T=0) and after 6 hours (T=6) of incubation.
We obtained the following data:
Table 4: Absorbance at 600 nm measured at T=0 hours and T=6 hours for two different colonies for each test device and a blank (LB+Chlor). Each colony had four technical replicates (Replicate 1-4).
The absorbance measurements show growth of all bacterial cultures carrying different devices when comparing T=0 hours and T=6 hours. There is a difference in growth between for cultures carrying different devices, but within cultures carrying the same device only little variation is observed (for both colonies and technical replicates). As expected, the blank (LB+Chlor) shows no growth as no cells should be present in this sample.
3.3 Fluorescence
Fluorescence (excitation: 485 nm, emission: 525 nm) was measured for two colonies for each device. For each of the colonies, four technical replicates were measured. The measurement was performed at the immediate start of the incubation (T=0) and after 6 hours (T=6) of incubation.
We obtained the following data:
Table 5: Fluorescence (excitation: 485 nm, emission: 525 nm) was measured at T=0 hours and T=6 hours for two different colonies for each test device and a blank (LB+Chlor). Each colony had four technical replicates (Replicate 1-4).
Similar to the absorbance measurements, the fluorescence measurements show an increase of fluorescence for all of the bacterial cultures carrying different devices when comparing T=0 hours and T=6 hours. There are differences in fluorescence between cultures carrying each of the devices, but within each device there is only slight variations (for both colonies and technical replicates). The blank (LB+Chlor) shows no increase of fluorescence as no cells and thus no fluorescence reporter is be present in this sample.
4. Colony Forming Units per 0.1 OD600 E. coli cultures
In this experiment, E. coli transformed with the following plasmids were used:
- Negative control BBa_R0040 Kit Plate 7 Well 2D
- Positive control BBa_I20270 Kit Plate 7 Well 2B
Two colonies were selected after each transformation and used for overnight cultures.
Step one: Starting sampling preparation
The overnight cultures were diluted and the OD600 was measured. Using this data, the solution was further diluted to an OD600=0.1. This was done in triplicate for each culture. It was measured for all samples whether an OD600=0.1 was obtained. The following OD600 was measured (supposed to be 0.1):
Table 6: The measured OD600 for each of the technical triplicates for each of the two colonies of the positive control and negative control. The measurement was made to confirm that an OD600=0.1 was obtained after dilution of the cultures.
Step two: Dilution series
A dilution series was then made using with the starting samples. 100 µl of dilution number 3, 4 and 5 were plated and grown overnight. 100 µl of dilution number 3, 4 and 5 were plated on LB agar plates with appropriate antibiotics and grown overnight. The final dilution factor for the three used dilution were as follows:
- Dilution 3: 8*104
- Dilution 4: 8*105
- Dilution 5: 8*106
Step three: CFU/mL/OD Calculation Instructions
The colonies on the plates were counted and CFU per 1mL of an OD600 = 0.1 culture were calculated. We obtained the following data:
Table 7: The counted number of colonies for each of the technical triplicates for each of the two colonies of the positive control and negative control.
The plates with colonies carrying one of the two different devices show less and less colonies for each dilution. A 10-fold reduction in the number of colonies was expected for each of the 10-fold dilutions, but this was only observed for some of the dilutions. The number of colonies within the technical triplicates are roughly similar although some outliers are present.
Table 8: The calculated number of colonies (pr. mL in the OD600 starting sample) for each of the technical triplicates for each of the two colonies of the positive control and negative control. The calculation was performed by multiplying the number of colony with the final dilution factor.
Note that because no colonies are present on “positive 2, dilution 5, triplicate 2” and “negative 1, dilution 5, triplicate 2” (table 7), they will appear as having no cells present (table 8).
5. Final comments
The data was submitted to the measurement committee the 24th of July and was accepted by the measurement committee the 30th of July.
Completing the InterLab studies served as a good introduction for us to be self reliant in the laboratory and train basic laboratory skills. Moreover, we learned our way around the laboratory and our facilities before starting the project.
We think that the idea behind the InterLab study is great and we look forward to see the other groups’ results and the final conclusion of the collected data from the iGEM HQ.