Team:Munich/InterLab

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InterLab Study We took part in the Fourth International InterLab Measurement Study which aims to stablish a protocol for GFP measurement in a plate reader based on engineering principles. To determine how similar the measurements can be in different labs, participating iGEM teams had to measure the fluorescence from 6 RBS devices in a plate reader. Why plate readers? With plate readers, the path length depends on the volume of the sample because the light goes through vertically. Instead, in a spectrophotometer, the light goes through horizontally and it is usually standardized to be 1 cm. Plate. Cuvette Materials Plate reader: CLARIOstar (BMG LABTECH) Plate reader plates: IBIDI96 (black plates with clear flat bottom) Cell culture shaker: Innova44 Devices: Positive control: BBa_I20270 Negative control: BBa_R0040 Device 1: BBa_J364000 Device 2: BBa_J364001 Device 3: BBa_J364002 Device 4: BBa_J364003 Device 5: BBa_J364004 Device 6: BBa_J364005 Calibration material: LUDOX for absorbance and Fluorescein for fluorescence (provided in the IGEM distribution kit) Microorganism: Escherichia coli DH5⍺ strains Methods We followed the protocol provided by iGEM HQ. Comments: We used the plasmids from the Plate 7 for all the devices except Device 1, which was taken from Plate 6. We performed both chemical transformation and electroporation for all devices to be sure we had enough colonies for the experiments. Plate reader configuration: Gain: 600 Number of flashes per well (absorbance): 22 Number of flashes per well (fluorescence):: 40 Temperature: 37 °C Filter: Dichroic filter 491.2 nm Emission wavelength: 515-20 nm Excitation wavelength: 470-15 nm Fluorescence reading: bottom optics Plate reader picture Results and Discussion: Device 1 from Plate 7 was problematic to transform. We obtained no colonies after trying several times chemical transformation and electroporation, so we think there was some problem with that specific well. Then, we used the Device 1 from Plate 6, and we obtained several colonies that we could use for the InterLab. Later, we found that in the Registry of Standard Biological Parts website, it is stated that Device 1 is a "complicated sample", so this could mean that there is some inherent problem with the device itself. Most devices showed a similar growth behavior except Device 1, which had the lowest growth rate. In terms of fluorescence, the Device 2 produced the highest amount of fluorescence, even more than the positive control, followed by Device 4. The Device 3 and 6 produced no fluorescence. Device 1 and 5 produced very little fluorescence after 6 hours. Since all the transformed bacteria showed very similar growth patterns across the devices, the lack of fluorescence can be attributed to a problem in the device itself or a transformation problem. Regarding the plate reader measurements, bubble formation was problematic because it led to wrong measurements or outliers in our data. We solved this by checking for bubbles before measuring and we set the plate reader to lightly shake the plate while measuring the data. Also, we did three measurements per plate to minimize the outliers. Fluorescence calibration was done by measuring Fluorescein at different concentrations. Absorbance (OD = 600 nm) raw values at different time points of all devices. Fluorescence (470/515 nm) raw values at different time points of all devices. Conclusion Device 2 showed the best fluorescence results, even better than the positive control. Device 4 was the second one with the highest emission. Device 1 was problematic from the beginning, so probably the low growth rate and low fluorescence emission was caused by a problem with the plasmid sequence. Device 5 also emitted low fluorescence, and device 3 and 6 showed no fluorescence.