The ability for parts to provide repeatable outputs is key to engineering applications, including design aspects found in synthetic biology. The purpose of the 2017 InterLab study was to address how reproducible outputs from standardized parts can be in different lab settings. The study also addressed what levels of protein expression could be driven by standardized promoters and ribosomal binding sites (RBS). Constructs were provided by the iGEM measurement committee which utilized green fluorescent protein as a reporter. This allowed the InterLab study contributors to easily follow protein expression driven by these standardized parts.
In order to better compare expression levels between research groups, the 2017 InterLab study incorporated calibration protocols to allow for reporting of fluorescence in absolute levels. Specifically, fluorescence levels from biological samples were calibrated against a fluorescein standard curve. Therefore, the resulting fluorescence results are reported in terms of the amount of fluorescence produced by a certain concentration of fluorescein.
The negative control for the InterLab study did not contain DNA coding for recombinant fluorescent-protein expression, thus this was well-suited for examining and accounting for background fluorescence of cellular components. The remaining constructs contained GFP (E0040) under the control of various promoters and ribosomal binding sites. The promoters and ribosomal binding sites utilized for Test Devices 1-6 are outlined below. All constructs contained E0040 (GFP protein sequence), B0010 (transcription terminator T1 rrnB from E.coli), and B0012 (transcription terminator TE from coliphage T7) following the promoter and ribosomal binding site.
|Test Device 1||BBa_J364000||J23101||B0034|
|Test Device 2||BBa_J364001||J23106||B0034|
|Test Device 3||BBa_J364002||J23117||B0034|
|Test Device 4||BBa_J364003||J23101||J364100|
|Test Device 5||BBa_J364004||J23106||J364100|
|Test Device 6||BBa_J364005||J23117||J364100|
All methods were based on the iGEM InterLab 2017 Plate Reader Protocol found here:
The calibration protocol for the OD 600 point was performed, followed by the generation of a fluorescein fluorescence standard curve. More details of calibration protocols can be found here:
Following the iGEM InterLab protocol, the eight plasmids, Positive Control, Negative Control and Test Device 1-6, were transformed into E. coli DH5α cells.
Two colonies were picked from each sample plate and grown overnight in 5mL of LB + Chloramphenicol at 37ºC and 220 rpm.
According to the provided InterLab study protocol, the OD600 and fluorescence were measured (excitation 485 nm, emission 530 nm) by our Microplate reader(SpectraMax® i3x Multi-Mode Detection Platform) at 0, 2, 4 and 6 hours. Clear plates for reading the samples were used. Full details of the protocol can be found here:
Calibration: Fluorescein Standard Curve
The SpectraMax® plate reader utilized for our data displayed a fluorescein-dependent increase in fluorescence. The data was plotted with both a linear, and a log scale x-axis (Figure 1). At very high fluorescein concentrations (25 and 50 μM), the data displayed a non-linear trend when plotted with a linear x-axis (Figure 1A), likely due to over saturation of the detector. However, the remaining details of the calibration curves are consistent with the result desired by the iGEM InterLab committee (values forming a straight line on linear and log scale x-axis, 1:1 slope, etc.).
Figure 1 - Fluorescein Calibration Curves showing the emission (530nm) due to the concentration of fluorophore in solution. A Linear x-axis. B Log scale x-axis.
Expression of Fluorescent Proteins from Test Devices
The Cell growth and fluorescent protein expression from the six test devices and the two controls, displayed varying levels of μM Fluorescein/ OD600 (Figure 2). As expected, the negative control construct exhibited little background fluorescence for both biological replicates. The positive control construct exhibited reproducible patterns of fluorescent protein expression over the course of the experiment, with μM Fluorescein/ OD600 peaking at the two hour time points for both biological replicates. Test Device 1 exhibited variable, but high μM Fluorescein/ OD600 values throughout the course of the experiment. While Test Device 2 exhibited mid-range levels of μM Fluorescein/ OD600 values, peaking at the two hour time point. Test Device 3 exhibited background levels of μM Fluorescein/ OD600 throughout the course of the experiment. Test Device 4 displayed high-levels of μM Fluorescein/ OD600, with the highest expression levels occurring from 2 hours onward. Test Device 5 displayed low, but above background levels of fluorescence with μM Fluorescein/ OD600 peaking at the 2 hour time point, whereas Test Device 6 displayed only background levels of μM Fluorescein/ OD600.
Figure 2 - Fluorescent Protein Expression of Test Devices. Fluorescent and OD600 measurements obtained at 0, 2, 4 and 6 hours represented as a bar graph in μM Fluorescein/ OD600 for the two controls and 6 test devices.
The lowest levels of fluorescent protein expression came from Test Devices 3 and 6. These devices both utilized the J23117 promoter, which had been previously characterized as being the weakest-strength of the promoters tested in the 2017 InterLab study (see J23100-J23119 on the registry of standard biological parts: http://parts.igem.org/Part:BBa_J23117). In terms of the remaining two promoters, constructs utilizing J23101 displayed higher fluorescence levels than constructs utilizing J23106. Because of this, we claim the relative strength of the promoters is J23101>J23106>J23117.
When comparing Test Device 2 to Test Device 5, it is clear that the B0034 ribosomal binding site utilized by Test Device 2 elicits higher levels of protein expression than the J634100 ribosomal binding site utilized by Test Device 5. As both devices contained the same promoter, differences in protein expression levels are attributed to differences in the ribosomal binding site. This trend of B0034 driving higher protein expression than J634100 continues when comparing Test Devices 1 and 3, where Test Device 1 contains B0034. Because of this, we therefore claim that B0034 drives protein expression at higher levels than J634100. In terms of absolute expression values (in terms of μM Fluorescein/ OD600), our results will be compiled with teams who completed the InterLab study, and analysis on how reproducible these absolute expression levels are among labs worldwide will be conducted.