Part:BBa_K2448035:Experience

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Applications of BBa_K2448035

Screening protocol

E. coli cell were transformed either with this part or with it's Dpe mutated version. All the clones present on the plates after the transformation were cultivated and the psicose concentration was evaluated using the protocol described for BBa_K2448025 but in the presence of 50 g/L of fructose per well for 9 to 10 hours as this is the optimal measurement time according to our biosensor characterisation. All tests were performed in technical duplicates. Fluorescence measurements (mCherry) have been normalized on cell density (OD600).

Our Application

Screening of a library of mutants of our enzyme, the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021) using this part (and it's mutant derivatives) allowed us to:

• show that our screening process works under realistic conditions. The biosensor we developed is able to give accurate assessment of the psicose production on positive control clones carrying the wild-type enzyme, but also on bacteria carrying mutant versions of Dpe. According to raw data, fluorescence of the positive control (WT, BBa_K2448035) is around 4500 arbitrary units. Biosensor characterization allowed us to correlate fluorescence and psicose concentration. We can estimate that psicose produced by the enzyme in WT is around 10 grams per liter which is coherent with measurements perform with BBa_K2448033.
• This screening process also allowed us to screen around 400 mutants and discover at least 2 enzymes with potentially improved activity (>30%) according to the fluorescence data. Four other variants displayed potentially improved activity, but of less then 10%.

User Reviews

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