Manufacturing

The Market

Figure 3 - Time evolution of the relative fluorescence per cell, normalized by the LB+Cam activity. All colonies are in their log-phase, suitable for evaluating their promoter strengths (see below). Notice that for all times, Device 1 is stronger than Device 2, while Device 3 is by far the weaker.

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Cytometry

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Negative control (no plasmid)

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Negative control (with plasmid)

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Positive control

For comparaison purposes, a positive control expressing GFP (BBa_I20270 - a GFP expression device in the pSB1C3 backbone (chloramphenicol resistant) was also measured.

Device 1

Device 2

Device 3

Comparison of expression rates of J23101, J23106 and J23117

Using flow cytometry we also conclude that promoter strength decreases from J23101>J23106>J23117 (Device 1>Device2>Device3).

Relative Promoter Units

According to the Anderson Promoter Collection [1], Device 1 should be stronger among the tested promoters in this experiment. Moreover, we can numerically measure a relative promoter strength and compare with the values available in the Anderson Collection. To measure then, we have to evaluate the following equation while our colonies are in the log-phase of their growth: \[ RPU_{\phi} = \frac{ \left\langle \frac{dF_{\phi}}{dt}\frac{1}{OD_{\phi}} \right\rangle }{ \left\langle \frac{dF_{J101}}{dt}\frac{1}{OD_{J101}} \right\rangle } \]

Measuring Promoter Strength using a Camera + Gimp

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Discussions

Our findings were consistent with the reported Anderson promoters strength, J23101 exhibited the stronger promoter activity followed by J23106 and J23117 was the weakest promoter and this result being confirmed fluorescence reading on a plate reader and flow cytometer. Additionally, using the plate reader we measured fluorescence over time (12 hours) at room temperature.

Protocols

Assemble protocol

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Sample preparation protocols

E. coli DH5α cells transformed with plasmids containing device 1, device 2 and device 3 were plated on LB agar (SIGMA) plates supplemented with chloramphenicol and grown for 18-20 hours at 37°C. Into independent sterile tubes with 5 ml of LB media (1:4) containing 34 μg mL^-1 of chloramphenicol, three colonies of each device and control were tip from the LB agar plates and grown at 37 °C, 80 rpm overnight ( for 14-16 hours). All tests were performed in E. coli DH5α cells in high-copy plasmid pSB1C3.

Plate reader

Samples were prepared in a black, flat-bottomed, 96-well Costar plate by adding a volume of 200 µL per well of each sample (180 µL of LB media and 20 µL of pre-inoculum). We also measured optical density using a clear, round-bottomed, 96-well Greiner plate

Flow cytometer

Cultured bacteria were diluted to approximately 8 x 10⁶ bacteria mL^-1 or OD₆₀₀ of ~ 0.01 (cell concentration must be in the range of 1x10⁶ to 2 x 10⁷ bacteria mL^-1 according to the equipment manufacturer’s recommendation). At this concentration, we guarantee enough cells in the samples and avoid clusters formation. To reduce background fluorescence originated from components of the media, samples were washed twice with phosphate buffer saline (PBS) at centrifugation 5000 g for 5 minutes. The supernatant was removed and the pellet resuspended in 1 ml sheath fluid, same volume as used to obtain OD₆₀₀ of 0.01.

Data acquisition protocol

Plate reader

Data acquisition was performed in a plate reader model SpectraMax® M3 (Molecular Devices), SoftMax Pro software. To prevent cross-talk and light scattering, samples were prepared in a black plate and top read was set to acquire fluorescence measurements.

Flow cytometer

Flow cytometry measurements were acquired in a BD FACSCaliburTM Flow Cytometer (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA) equipped with BD FACSComp™ and BD CellQuest™ Pro software. First the equipment was calibrated to ensure optimal performance due to inherent instrument variability. As model calibrators we used BD Calibrite 3 Beads (BD Calibrite™) by running FACSComp program.

For each sample, ten thousand events were acquired and fluorescence was measured in FL1 and FL2 channels. Voltage and amplifier gain were adjusted in the Detector/Amps window according to negative control (no plasmid) (Figure negative control no plasmid) to ensure all the events are in the plot FSC vs. SSC and the same parameters were used to read GFP expressing cells and other controls. FL1 vs. FL2 plot was divided in 4 quadrants, and the lower left quadrant (LL) contained all events from the negative controls. The lower right quadrant (LR) should show up the GFP expressing cells and histogram FL1 vs. counts, the fluorescence levels. Although, the histogram do not represents the origin of fluorescence and populations, then FL1 vs. FL2 plot can show the origin of the fluorescence.