The characterization of standard parts has always been one of the main concerns in Synthetic Biology. For this very same reason, iGEM teams from all around the World were suggested to take part in the biggest measurement study ever conducted and the 2015 UNITN-Trento iGEM team answered the call. The goal of this Second International Measurement Interlab Study is to assemble three different devices, each one containing a promoter with a screening plasmid intermediate and collect as many fluorescence data as possible. iGEM teams are free to use any technique to measure their devices as long the obtained data are solid and reproducible.
We used the three mandatory devices for the measurement study:
- Device 1: BBa_J23101 + BBa_I13504 in pSB1C3
- Device 2: BBa_J23106 + BBa_I13504 in pSB1C3
- Device 3: BBa_J23117 + BBa_I13504 in pSB1C3
- Negative control: BBa_R0040 in pSB1C3
- Positive control: BBa_I20270 in pSB1C3.
The measurement devices were prepared by amplifying the reporter (BBa_I20270) by PCR. The amplified insert was then cut with XbaI and PstI and ligated into the plasmid containing the promoter previously cut with SpEI and PstI. All the devices were confirmed by restriction digestion as well as DNA sequencing.
The confirmed devices were then transformed in different bacterial strains of E. coli: NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorometer, and BD FACSCanto flow cytometer.
We also focused on transcription since the characterization is about promoters. To do so we performed RT-qPCR using a BioRad CFX96 Touch™ Real-Time PCR Detection System. Additionally, we performed an in vitro characterization study, by measuring the fluorescence intensities of each device with a Cell-Free E. coli S30 Extract System with a Qiagen Rotor-Gene Q as the spectrofluorometer.
Extraction from the Registry
Polymerase Chain Reaction (PCR)
Restriction Digestion of plasmids containing the promoter
Glycerol stocks preparation and Sample Growth
Fluorescence readings: Tecan INFINITE 200 PRO Plate Reader
Fluorescence readings: Cary Eclipse Fluorescence Spectrophotometer
Fluorescence readings: BioRad CFX96 Touch Real-Time PCR Detection System
Fluorescence readings: BD FACSCanto Flow Cytometer
Fluorescence readings: E. coli S30 Extract System for DNA Circular
Our characterization confirmed the relative strength of the promoters
J23101 is the strongest promoter among the three, showing high expression of GFP in all three strains, regardless of the technique used. J23106 is the medium promoter and J23117 the weakest.
Ratios across promoters are kept the same
J23101/J23106 fluorescence ratios ranged from 2.0 to 4.5, depending on the strain and the technique. Differently from the other two promoters, J23117 showed a very low GFP production, as it was not detectable by eye or using the trans-illuminator and showed little fluorescence with the three techniques used.
Different techniques lead to the same results, with different sensitivities
The best way to perform a characterization is to use various techniques. Throughout our experiments we saw that each instrument has a specific sensitivity, which alters the output data. The FACS happened to be the most accurate among all, due to its extremely high intrinsic sensitivity. The plate reader also showed a good accuracy while the fluorometer was not able to detect the weakest promoter from the background noise. The use of the qPCR machine as a spectrofluorometer also gave positive results.
In vitro conditions mimic the in vivo reality
Comparing the results obtained from the cell-free extract to the others, we discovered that the promoters behave the same when working in vitro or in living bacteria. Since testing constructs in vitro is much faster than in vivo, our results suggest that it may be wise to first screen parts and/or genetic circuitry in vitro. Then the activity of a smaller subset could be confirmed with in vivo measurements.
Looking at promoters from a different angle
In vitro characterization by qPCR allows for the quantification of promoter strength by measuring RNA transcript levels, rather than by looking at the concentrations of something one step removed, i.e. protein. This approach gives a a more direct measure of promoter strength.
Bacterial strain does matter
The three promoters behaved differently in the different bacterial strains used. The bacterial strain which gave the highest fluorescence was NEB10β, which showed significantly increased expression of protein in all cases when compared with JM109 and NEB Express. Clearly this discordance among strains must to be due to their different genotypes. A different bacterial proteome (e.g. presence/lack of specific proteases and/or chaperonins) may alter protein production, processing and folding, and thus fluorescence emission.