Development of a high-throughput screening method for recombinant Escherichia coli with intracellular dextransucrase activity.

So-Ra Lee, Ah-Rum Yi, Hong-Gyun Lee, Myoung-Uoon Jang, Jung-Mi Park, Nam Soo Han, Tae-Jip Kim

Index: J. Microbiol. 49(2) , 320-3, (2011)

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Abstract

To efficiently engineer intracellular dextransucrase (DSase) expression in Escherichia coli, a high-throughput screening method was developed based on the polymer-forming activity of the enzyme. Recombinant E. coli containing the Leuconostoc citreum DSase (LcDS) gene was grown on Luria-Bertani agar plates, containing 2% sucrose, at 37°C for 8 h. The plates were then evenly overlaid with 0.6% soft agar, containing 1.2 mg/ml D-cycloserine, and incubated at 30°C to allow gradual cell disruption until a dextran polymer grew through the overlaid layer. A significant correlation between dextran size and enzyme activity was established and applied for screening truncated mutants with LcDS activity.