Fifth International InterLab Measurement Study
Introduction
The InterLab Study is an international effort to identify and eliminate the sources of systematic variability in synthetic biology measurements. Teams from around the world collaborate to build robust measurement procedures that would ensure accurate comparison between data obtained from different labs. The measurement marker used in InterLab is the Green Fluorescent Protein (GFP), expressed in E. coli. The main source of variability that this year’s Study aims to eliminate concerns the number of cells in the sample when bulk measurements of a cell population are taken. For this reason, in order for the total fluorescence to be comparable, it has to be divided by the number of cells. The number of cells is usually related to absorbance of light in 600nm and optical density (OD) of the sample but it is not clear if this is a reliable method. Therefore, the main question that the Fifth InterLab Study poses is:
This year’s Study discusses data from six different GFP expression plasmids, a positive and a negative control and is conducted according to iGEM’s protocols.
Materials & Methods
Devices: BBa_R0040 (Negative Control), BBa_I20270 (Positive Control), BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009.
Plasmid Backbone: pSB1C3.
The devices were transformed in Escherichia coli K-12 DH5 α. Two colonies of each of the transformed cells were grown overnight in 10mL LB media and chloramphenicol (20μg/mL) at 37oC, 220rpm. Cultures were diluted to reach Abs600 of 0,02 in a total volume of 12mL LB with Chloramphenicol.
Abs600 Measurement
At 0 hours and 6 hours 4 replicates of 100μL of the diluted cultures were placed in transparent, flat bottom 96-well plates. Absorbance of LB with Chloramphenicol was also measured as control sample. The absorbance of was measured in Spectra Max 250 (Molecular Devices) SPECTROstar Nano Microplate Reader (BMG Labtech), with disabled pathlength correction. Before the measurement, the system was calibrated by setting the OD reference point using LUDOX CL-X and then by constructing a standard curve of particle concentration using Silica Beads.
Fluorescence Measurement
As the plate reader available at National Technical University of Athens does not measure fluorescence, a different plate reader was used for this measurement. The cells were transferred in a closed thermo-insulated box filled with ice, to the Agricultural University of Athens, where the fluorescence was measured in a VICTOR X2 Multilabel Plate Reader (PerkinElmer), for the samples at 0 and 6 hours. First, a fluorescence standard curve was constructed, using fluorescein in PBS.
Fluorescence was also measured for two of the four replicates of each part with Flow Cytometry, in a BD FACSCanto II, using Rainbow beads by SpheroTech for calibration (catalog number: RCP-30-5Α).
Colony Forming Units(CFUs) per 0.1 OD600 E. coli cultures
Starting samples of OD600=0.1 were prepared in triplicates from overnight cultures of the Positive Control (BBa_I20270) and the Negative Control (BBa_R0040). Five serial dilutions were performed and for the dilution factors 8E+04, 8E+05 and 8E+06, 100μL were aseptically spreaded on plates with LB media and Chloramphenicol and incubated at 37oC overnight. The number of colonies was counted and assuming that one bacterial cell gives rise to one colony, CFU per 1mL of culture was calculated.
Protocols:
Results
Discussion
As already mentioned, the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells. In order to determine the mean expression level of GFP per cell, we need to divide the total fluorescence by the number of cells.
This is usually done by measuring the absorbance of light at 600nm, from which the “optical density (OD)” of the sample is computed, as an approximation of the number of cells. However, OD measurements are subject to high variability between labs and it is unclear how good of an approximation an OD measurement actually is. As far as our OD measurements are concerned, we noted that at 0 hours the absorbance values were very low as the cell cultures had not had the time to grow. For this reason, there were a few invalid measurements that lead to div/0 or negative values and that were excluded from the above graphs.
In order to remove this source of variability in our measurements, we used a more direct method to determine the cell count in each sample, namely CFU measurements. Having determined the cell count in each sample both using the traditional OD measurements and using CFUs, we hope that the results of our efforts will prove helpful and the iGEM foundation will draw meaningful conclusions using them, combined with the results of all the other teams that took part in the Interlab study.
Last, we would like to note that the main difficulty in carrying out this experiment was finding the instruments needed. We had to transfer the cultures in three different universities across the city, and given the hot climate in Athens during the summer, our measurements might have been slightly affected, although we took all possible precautions.
Acknowledgements
Technical support
- Anastasia Zerva, School of Chemical Engineering, National Technical University of Athens
- Antonios E. Koutelidakis, Department of Food Science and Nutrition, University of the Aegean
- Ioannis V. Kostopoulos, Department of Biology, National and Kapodistrian University of Athens
- Ourania E. Tsitsilonis, Department of Biology, National and Kapodistrian University of Athens
Instrument provision
- Maria Kapsokefalou, Department of Food Science and Human Nutrition, Agricultural University of Athens
- Ourania E. Tsitsilonis, Department of Biology, National and Kapodistrian University of Athens
