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KMID : 0880220110490020320
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Journal of Microbiology
2011 Volume.49 No. 2 p.320 ~ p.320
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Development of a high-throughput screening method for recombinant Escherichia coli with intracellular dextransucrase activity
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Lee So-Ra
Yi Ah-Rum
Lee Hong-Gyun
Jang Myoung-Uoon
Park Jung-Mi
Han Nam-Soo
Kim Tae-Jip
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Abstract
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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.
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KEYWORD
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high-throughput screening, D-cycloserine, dextransucrase, recombinant E. coli
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