Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening.
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-1b) was quantitatively characterized under various growth temperatures, heat induction temperatures and durations, and IPTG (isopropyl beta-d-thiogalactoside) induction times, after resolving its erratic lysis profile found previously. Escherichia coli BL21 cells harboring this vector grew well at temperatures <36 degrees C, and lysed efficiently (97.0 +/- 0.8%) just 0.5 h after heat induction at 42 degrees C for 30 min when cell growth was performed at 35 degrees C. 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.
Cai
Zhen
Z
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.
Xu
Wanghui
W
Xue
Rui
R
Lin
Zhanglin
Z
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001232:Aspergillus fumigatus; D001433:Bacteriolysis; D002478:Cells, Cultured; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors; D006358:Hot Temperature; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D029968:Escherichia coli Proteins; D007544:Isopropyl Thiogalactoside; D000594:Amino Acid Oxidoreductases; C103012:amadoriase