Overview
The Interlab study is a unique part of the iGEM competition, which aims to develop robust measurement procedures for different types of calibrants. To achieve this, the Engineering Committee collects results from different labs worldwide and evaluates the variability and reproducibility of the measurements taken by different teams using the same protocol. The results are then published in international peer-reviewed journals, giving the participants the chance to participate in scientific publishing.
This year, the Interlab study wants to extend measurement procedures from one-colour calibrations (green) to multi-color calibrations (green-red-blue). In addition, not only one-colour but also dual-colour devices will be tested for repeatability. Finally, the study wants to analyse the repeatability of the production of green fluorescence over time when cells are cultivated in 96-well plates as opposed to culture tubes.
The Interlab Study is a great opportunity for iGEM teams to begin their lab journey and learn some useful lab techniques. As a team, we are interested in practising new techniques in our lab and at the same time cooperating with other teams from across the world to address the same problems. Moreover, we hope that the results and knowledge that we gained during these experiments will be helpful to future iGEM teams and contribute to iGEM’s general goals.
Protocols and general information
The experiment protocols were provided by the measurement committee. Competent DH5α cells were used (as recommended) for our experiments, which were transformed using the appropriate devices. The transformed bacteria were selected from agar plates containing chloramphenicol and then cultivated in LB medium that contained chloramphenicol according to the protocols. After 16 hours, the different cultures were transferred into black 96-well plates with a transparent flat bottom to be measured. To measure the absorbance and fluorescence, Promega Glomax multi-detection plate reader was used with the following settings:
- Absorbance: 600 nm
- Green fluorescence: excitation wavelength: 490.0 nm, emission filter: 510.0 - 570.0nm
- Red fluorescence: excitation wavelength: 525.0 nm, emission filter: 580.0 - 640.0nm
Calibration
Calibration tests were performed in order to create standard curves and be able to analyse and compare our data. Unfortunately, our team had an issue with the silica beads (Monodisperse Silica Nanoparticles), so we didn’t submit the results for this calibration. Here we present our standard curves for green and red fluorescence.
Figure 1: Green fluorescence measured at excitation wavelength: 490.0 nm, emission filter: 510.0 - 570.0nm for different concentrations of fluorescein (0 µM to 10 µM)
Figure 2: Red fluorescence measured at excitation wavelength: 525.0 nm, emission filter: 580.0 - 640.0nm for different concentrations of sulforhodamine 101 (0 µM to 10 µM)
Results
Experiment 1
In this experiment, we tested the fluorescence of devices that either encode one fluorescence protein (blue, green, or red), or two fluorescence proteins. The aim of these measurements was to examine the lab-to-lab reproducibility of the new three-colour calibration protocol.
Transformation of E. coli DH5 alpha cells was successful for all of 8 devices (used devices are available in the iGEM protocol for experiment 1). After appropriate cultivation, absorbance and fluorescence were measured in 200 ul of every culture at 0 and 6 hours. The results are presented in the following diagrams.
OD600
Figure 3: Absorbance of transformed cells after 0h cultivation (600 nm)
Figure 4: Absorbance of transformed cells after 6h cultivation (600 nm)
Green Fluorescence
Figure 5: Measurements of green fluorescence in transformed cells after 0h cultivation (excitation: 490.0nm, emission filter: 510.0 - 570.0nm)
Figure 6: Measurements of green fluorescence in transformed cells after 6h cultivation
(excitation: 490.0 nm, emission filter: 510.0 - 570.0nm)
Red Fluorescence
Figure 7: Measurements of red fluorescence in transformed cells after 0h cultivation
(excitation: 525.0 nm, emission filter: 580.0 - 640.0nm)
Figure 8: Measurements of red fluorescence in transformed cells after 6h cultivation (excitation: 525.0 nm, emission filter: 580.0 - 640.0nm
After the measurement of the green and red fluorescence in our bacterial cultures, we believe that the experiment was successful. After cultivation for 6 hours, we can observe bacterial growth and, at the same time, increase of the fluorescence in the bacterial culture that were transformed with green or red devices (for example test device 1 & 2).
Experiment 2
In this experiment, we tested the fluorescence of six devices that encode two fluorescence proteins in two transcriptional units. Some of the devices differ only in the order of the encoded transcriptional units. The aim of these measurements was to examine the lab-to-lab reproducibility of the three-colour calibration protocol when two fluorescent proteins are expressed in the same cell.
Transformation of E. coli DH5 alpha cells was successful for all of 8 devices (used devices are available in the iGEM protocol for experiment 2). After appropriate cultivation, absorbance and fluorescence were measured in 200 ul of every culture at 0 and 6 hours. The results are presented in the following diagrams.
OD600
Figure 9: Absorbance of transformed cells after 0h cultivation (600 nm)
Figure 10: Absorbance of transformed cells after 6h cultivation (600 nm)
Green Fluorescence
Figure 11: Measurements of green fluorescence in transformed cells after 0h cultivation (excitation: 490.0 nm, emission filter: 510.0 - 570.0nm)
Figure 12: Measurements of green fluorescence in transformed cells after 6h cultivation (excitation: 490.0 nm, emission filter: 510.0 - 570.0nm)
Red Fluorescence
Figure 13: Measurements of red fluorescence in transformed cells after 0h cultivation (excitation: 525.0 nm, emission filter: 580.0 - 640.0nm)
Figure 14: Measurements of red fluorescence in transformed cells after 6h cultivation (excitation: 525.0 nm, emission filter: 580.0 - 640.0nm)
In the second interlab experiment our results were ambiguous. Results of green fluorescence show that the order of the encoded transcriptional units is irrelevant to the expression of the protein and the fluorescence. On the contrary, results of red fluorescence show that when the red transcriptional units are first, ρrotein is produced at higher levels and gives greater fluorescence levels.
Experiment 3
In this experiment, we tested the difference in fluorescence and absorbance when the bacterial cultures were incubated in 96-well plates instead of test tubes for culturing. The aim of the experiment was to evaluate how the plate culturing protocol performs in comparison to culturing in test tubes (such as a 50 mL falcon tube).
Transformation of E. coli DH5 alpha cells was successful for all of 8 devices (used devices are available in the iGEM protocol for experiment 3). After appropriate cultivation, absorbance and fluorescence were measured in 200 ul of every culture at 0 and 6 hours. The results are presented in the following diagrams.
OD600
Figure 15: Absorbance of transformed cells after 0h cultivation (600 nm)
Figure 16: Absorbance of transformed cells after 6h cultivation in 96 well plate (600 nm)
Figure 17: Absorbance of transformed cells after 6h cultivation in tube culturing (600 nm)
Green Fluorescence
Figure 18: Measurements of green fluorescence in transformed cells after 0h cultivation (excitation: 490.0 nm, emission filter: 510.0 - 570.0nm)
Figure 19: Measurements of green fluorescence in transformed cells after 6h cultivation in 96 well plate (excitation: 490.0 nm, emission filter: 510.0 - 570.0nm)
Figure 20: Measurements of green fluorescence in transformed cells after 6h cultivation in tube culturing (excitation: 490.0 nm, emission filter: 510.0 - 570.0nm)
The concentration of bacterial cultures seems to be higher in cultures that were cultivated in test tubes. In some cases (for example, in positive control green), the concentration of the test tube culture is twice that of the 96-well plate culture. In addition, the fluorescence measurements are also increased in bacterial cultures that are cultivated in test tubes.
Summary
The InterLab study was a unique and educational experience. We had the chance to train basic laboratory skills, to troubleshoot a lot of bacterial transformations and to become familiar with our laboratory and facilities before starting the project. We love the idea behind these studies and we are happy that we are able to contribute with our work. We look forward to the final results.
