Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

Abstract

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.

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

“…Generally, the extracellular level of ␤-galactosidase, a native cytoplasmic protein, is considered an indication of cell lysis (24,25). In the present study, it was found that, at 22 h postinduction, the percentages of ␤-galactosidase activity in the culture medium were similar in cells expressing cutinase NS , cells expressing inactive cutinase NS S130A, and control cells.…”

“…Cell lysis was determined as the percentage of extracellular enzyme activity versus the sum of extracellular and intracellular activities, using ␤-galactosidase as the reporter protein (24,25). Standard ␤-galactosidase activity was assayed according to the previously reported method (26).…”

“…␤-Galactosidase is a cytoplasmic protein, and under normal conditions, its level in the culture medium is very low; thus, the presence of extracellular ␤-galactosidase activity would be an indication of cell lysis (24,25). In the present study, at 22 h postinduction, 90.9% of the cutinase NS was found in the culture medium of E. coli cells harboring pET20b(ϩ)/cutinase NS , while only 7.2% of the ␤-galactosidase activity was found in the culture medium.…”

A novel cell autolysis system for cost-competitive downstream processing

“…Cloning junctions and cloned sequences were confirmed by sequencing of isolated plasmid DNA (Aokeding, China; or alternatively Invitrogen, China). The Lambda lysis system comprising the SRRz genes was amplified from the plasmid pEAS-1a (Xu et al 2006;Cai et al 2008) using the primers P1 and P2 (all primer sequences are listed in Table S1) and integrated using Gibson assembly into the standard part plasmid pSEVA331 (Silva-Rocha et al 2013;Durante-Rodríguez et al 2014) which was linearised via amplification using primers P3 and P4. The mutation S107 M3L and a standard part ribosome binding site (BBa_B0029; Jason Kelly, iGEM2006_MIT group) were provided on primer P1, whereas 13 bp of the native ribosome binding site of the chloramphenicol acetyltransferase (CAT) resistance gene was left in place before its coding sequence (CDS).…”

“…While this approach is relatively straight-forward and great progress has been made in miniaturisation and maintaining control over potentially lethal leaky expression in such systems on the laboratory scale (Xu et al 2006;Cai et al 2008), practical applications are likely to fail in industrial settings due to the fact that autolysis needs to be induced during the very last process step, and cells grown to a high cell density are very poorly responsive to induction (Gefen et al 2014).…”