Part:BBa_K2448029:Experience

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Applications of BBa_K2448029

Protocol

Culture preparation:

We set a liquid culture of transformed bacteria in LB supplemented with 35 µg/mL chloramphenicol and incubated it overnight at 37°C under constant shaking at 200 rpm. The day after, a 100X dilution of this culture was done in LB supplemented with 35 µg/mL chloramphenicol and incubated at 37°C under constant shaking at 200 rpm for 1 hour. Then, 120 µL were dispatched per well according to the 96 well plate map (Figure 1).

Plate preparation:

The experiment was conducted in a 96 well plate, the COSTAR® 3603 from Corning Inc. These plates allowed us to characterize two different biosensors at a time. Before placing the culture in each well, the plate is filled according to the plate map (Figure 1) with 30 µL solution containing Psicose and IPTG at a 5X concentration (thus, upon addition of the 120µL of culture, the Psicose and the IPTG will be at the right final concentration). Immediately after complete loading, the plate is placed in the plate reader. Peripheral wells are filled with water to avoid evaporation during the incubation time.

Measurement:

To perform the measurements, we used a CLARIOstar® (BMG LABTECH) plate reader, kindly lent by the Institute of Systems and Synthetic Biology (Evry, France). The plate reader is programmed to assess the fluorescence at mCherry optimal wavelength (587 nm for excitation and 610 nm for emission) and OD600. Those two parameters are measured every 7 minutes for 150 cycles. The plate is incubated in the device at 37°C under constant shaking at 200 rpm.

All tests were performed in technical duplicates and biological triplicates. Fluorescence measurements (mCherry) have been normalized on cell density (OD600).

Raw data for this biosensor are available on our wiki.

Figure 1. Example of a plate map used for the biosensor characterization.

Our Applications

We characterised this biosensor and showed that it has a perfect foldchange in range of concentrations corresponding to bioproduction range (1 mM to 300 mM psicose), but it tends to saturate at high concentrations. This is after BBa_K2448025 and BBa_K2448027 the third best candidate for screening a library of mutants and identify the cells carrying an improved version of our enzyme, the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021).

User Reviews

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