Team:Lund/InterLab
InterLab
Introduction
InterLab is a worldwide laborative collaboration where repeatable and reliable results is of very high importance for comparability. In synthetic biology one of the most important signalling proteins is green fluorescent protein (GFP), which fluoresces when exposed to ultraviolet light. Each participating team expressed GFP under promoters of varying strength and compared their measurements to different calibrations.
Materials and Methods
The three different calibration methods were:1. The absorbance at 600 nm was measured for Ludox CL-X of different known concentrations in a plate reader, and OD600 measurements from a spectrophotometer were used to create a conversion factor between absorbance measured by the plate reader and OD600.
2. To obtain a standard curve showing the relationship between Abs600 in the plate reader and actual cell count, absorbance of silica beads of the same size as the E. coli cells were measured at different known concentrations.
3. The fluorescence intensity of the salt fluorescein, which has similar excitation and emission spectra as GFP, was used to make a standard curve. This standard curve was then used to convert GFP fluorescence measurements into equivalent concentrations of fluorescein, a value which can be used to compare data between different labs regardless of what equipment they use for their measurements.
All calibrations were done using flat bottomed black 96-well plates.
E. coli DH5-α was transformed with 8 plasmids, each containing different inserts. The only difference between the inserts was the promoter, and for the positive control also the ribosome binding site (Table 1). For full description of the parts visit this page and search for them as stated in Table 1, column “Biobrick”. The cells were incubated according to iGEM measurements protocol for plate readers. The cell density at 600 nm and fluorescence were measured both before and after the incubation.
| Test device | Biobrick | Promoter strength (a.u) | RBS |
|---|---|---|---|
| Negative control | BBa_R0040 | Medium | - |
| Positive control | BBa_I20270 | 256 | B0032 |
| 1 | BBa_J364000 | 1791 | B0034 |
| 2 | BBa_J364001 | 1185 | B0034 |
| 3 | BBa_J364002 | 162 | B0034 |
| 4 | BBa_J364007 | 547 | B0034 |
| 5 | BBa_J364008 | 1831 | B0034 |
| 6 | BBa_J364009 | 396 | B0034 |
Results
All the raw data is available here.
The results of calibration 1 gave a conversion factor between OD600 and Abs600 of 2.571. The results for calibration 2 can be seen in fig. 1. A quite linear trend was formed. How the fluorescence intensity of the fluorescein salt depended on concentration is shown in fig. 2. Also here a linear trend showed.
How the cell fluorescence varied before and after the 6 h incubation is shown in fig. 3. Here it was possible to observe an increase for all but the negative control and the 3rd test device.
Discussion
According to the promoter strengths, test device 1, 2 and 5 should fluoresce more than the positive control and test device 3 which are the weakest. 4 and 6 should be somewhere in between.
According to fig. 4, test device 4 was the highest, 2 and 5 medium, 1, 3 and 6 were lower and the controls were low. Thus, the results were not completely off, but matched the expected outcome to some degree. Possible sources of error might be not perfectly regulated cells when it comes to protein production and growth or that the stated strength of the promoters were not what they were in reality.
