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A set of UV-inducible autolytic vectors for high throughput screening

Paper ID Volume ID Publish Year Pages File Format Full-Text
25514 43578 2007 6 PDF Available
Title
A set of UV-inducible autolytic vectors for high throughput screening
Abstract

A high throughput screening scheme is often a prerequisite for directed evolution of enzymes or metagenomic analysis of DNA samples. For assaying intracellular enzymes of interest (e.g. when Escherichia coli is used), it requires cell lysis in many cases, chemical or enzymatic, which can be tedious and cost-consuming. In this study, a set of UV-inducible autolytic vectors was constructed to offer a simpler means of cell lysis that is free of additional liquid handling. The SRRz lysis gene cassette from bacteriophage Lambda was cloned downstream of a UV-inducible promoter, the recA promoter or the umuDC promoter, and further inserted into the backbone of pUC18, and transformed into E. coli BL21 cells. The SRRz expression and cell lysis was induced by UV irradiation. For both the recA and umuDC promoters, at 30 °C the lysis efficiency was found to be consistent and above 60% as measured using β-galactosidase as the reporter. However, at 37 °C the lysis profiles were found to be erratic. UV lysis in 96-well plates also produced consistent lysis results that were comparable to those obtained by lysozyme treatment, demonstrating the utility of these autolytic vectors in high throughput screening. This set of artificial SRRz autolysis units should be transferable to other vectors. Surprisingly, it was found that the E. coli BL21(DE3) was also partially disrupted under UV irradiation, with a lysis efficiency of 44.5% at 30 °C, and 22.5% at 37 °C.

Keywords
Directed evolution; High throughput screening; UV induction; Autolysis; SRRz lysis cassette
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A set of UV-inducible autolytic vectors for high throughput screening
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 127, Issue 4, 20 January 2007, Pages 647–652
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us