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《微生物与人类健康科技论坛论文汇编》2009年
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Facile,reagentless and in situ release of E.coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

【摘要】:正In an effect to broaden the application of the heat-inducible autolytic vector pUC18-cI857/p_R-SRRz-rrnB previously developed,a new vector pUC18-cI857/P_R(T41C)-SRRz-rrnB(pEAS-lb) was quantitatively characterized under various growth temperatures,heat induction temperatures and durations,and IPTG induction times,after resolving its erratic lysis profile found previously.E.coli BL21 cells harboring this vector grew well at temperatures below 36℃,and lysed efficiently(97.0%±0.8%) just 0.5 h after heat induction at 421 for 30 min when cell growth was performed at 351.Application of this autolytic vector either in 96-well plates,or on nitrocellulose membranes,or on agar plates led to facile,efficient and consistent release of intracellular recombinant enzymes (e.g.,a lysis efficiency of 91.8%±1.1%was obtained in 96-well plates).Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector.This reagentless and in situ cell lysis method has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection,and even for lysis of cells in the microarray format.
【分类号】:Q78

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