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.

Extraction from the Registry

Polymerase Chain Reaction (PCR)

Electrophoresis gel of the reporter gene The bright band corresponds to the part BBa_I13504. The part was amplified by Phusion PCR and digested with XbaI and PstI. We can see the expected band at around 875 bp.

The reporter gene (i.e. GFPmut3b from part BBa_I13504) was amplified using the Phusion polymerase from New England Biolabs and primers matching the prefix and suffix.

We used 50 ng of template with an annealing temperature of 59 °C and an extension time of 90 seconds. The PCR was confirmed by electrophoresis and subsequently purified with NucleoSpin Gel and PCR Clean-Up Kit from Macherey-Nigel. 125 ng of purified PCR were digested with 1 μl of XbaI and 1 μl of PstI overnight at 37°C. The following morning the sample was treated with 1 μl of DpnI for 2 hour at 37°C and the enzymes were deactivated for 20 min at 80 °C.

Restriction Digestion of plasmids containing the promoter

Ligation

	Plasmid: Insert = 1:3	Control
Vector	100 ng	100 ng
Insert	125 ng	-
10X Buffer	2 μl	2 μl
Buffer (Stock 10X)	5 μl	5 μl
T4 - DNA Ligase	2 μl	2 μl
Water	Up to 20 μl	Up to 20 μl

The ligation reactions were assembled and incubated at room temperature for 1 hour according to the table beside. Subsequently 10 μl of the ligation mixture were transformed and plated into LB-agar plates with the proper antibiotic resistance. The confirmed devices were transfected in NEB10β, JM109, NEB Express.

Cells expressing the devices These three plates contains E. coli NEB Express transformed with J23101 (top left), J23106 (top right), and J23117 (bottom). Interestingly, promoters' strength are detectable by eye, depending on the colonies' color.

Cells expressing the devices. These three plates contains E. coli NEB Express transformed with J23101 (top left), J23106 (top right), and J23117 (bottom).

Parts Confirmation

Correct clones were screened by restriction digestion and confirmed by sequencing:

Cell pellets at the UV-trans illuminator Pellets of overnight NEB10β cells cultures expressing the assembled devices. Pellet's colors confirmed the promoter strength, (from left to right) the strongest promoter J23101, the medium strength promoter J23106, the weak promoter J23117, the positive control I20270, the negative control R0040.

Electrophoresis gel of all devices We digested three colonies for each device with XbaI and PstI. The correct assembly is confirmed by the band at 910 pb.

• BBa_J23101+BBa_I13504

  [Sequencing Data]

• BBa_J23106+BBa_I13504

  [Sequencing Data]

• BBa_J23117 +BBa_I13504

  [Sequencing Data]

Glycerol stocks preparation and Sample Growth

Fluorescence readings: Tecan INFINITE 200 PRO Plate Reader

The cells were thawed and resuspended in 3 ml of PBS. An aliquot of 150 μl of each sample was placed into a White, Flat-bottomed, 96-well Costar Plate (code: 3917) and fluorescence intensities were taken with a Tecan Infinite 200 Pro Plate Reader (made in Switzerland). Excitation wavelength and emission wavelength were 395 nm and 509 nm, respectively. The gain was optimized at 70 V and kept constant for each sample. PBS was used as blank. To obtain technical replicates, fluorescence intensities were acquired for three aliquots of the same biological sample, keeping the same instrumental conditions. The raw data were adjusted for the blank value and the means across the replicates with their relative standard deviation were plotted.

Fluorescence readings: Cary Eclipse Fluorescence Spectrophotometer

The cells were thaw and resuspended in 3 ml of PBS. Each measurement was taken in a clear acrylic cuvette (REF 67.755) with a Cary Eclipse Fluorescence Spectrophotometer (made in USA). The blank was done with PBS. The excitation wavelenght used was 395 nm and the spectra were acquired between 420 nm to 650 nm. The gain was optimized at 775 V and kept the same throughout the measurement.

The fluorescence intensity of the maximum emission wavelenght (514 nm) was adjusted for the blank and the calculated means with relative standard deviations were plotted.

Fluorescence readings: BioRad CFX96 Touch Real-Time PCR Detection System

Fluorescence readings: BD FACSCanto Flow Cytometer

The cells were grown from glycerol stocks as described above. Differently from before when they reached the OD of 0.7 were not frozen, but were used immediately to measure fluorescence intensity. An aliquot of 5 μl of cells was diluted in 900 μl of PBS. The instrument used was a BD FACSCanto (made in USA) set with the following parameters:

• FSC gain: 525 V
• SSC gain: 403 V
• FITC gain: 510 V
• Flow rate: LOW
• Total number of events in P2: 10000

Those parameters allowed the instrument to process 900/1500 events per second. We analyzed the raw data and plot the means of three replicates with relative standard deviations.

Fluorescence spectra from flow cytometer analysis of the NEB10β strain The merge shown above represents the devices J23101 (white), J23106 (yellow), J23117 (blue), and the negative control R0040 (orange). Data were analyzed using Cyflogic 1.2.

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 sentivity. 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.

Promoter strength across strains and techniques Values presented are the average of three technical replicates for each biological sample (total of 9 replicates). Data normalized on the the maximum expression value of each technique. Heatmap calculated with R Project.

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. We hypothesized this discordance among strains 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, thus fluorescence emission.

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.

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.

Strain influence on promoters efficiency Values presented are the average of three technical replicates for each biological (total of 9 replicates). Data normalized on the the maximum expression value of each technique. Heatmap calculated with R Project.