A set of UV-inducible autolytic vectors for high throughput screening.
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 degrees C the lysis efficiency was found to be consistent and above 60% as measured using beta-galactosidase as the reporter. However, at 37 degrees 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 degrees C, and 22.5% at 37 degrees C.
Li
Shuang
S
Department of Chemical Engineering, Tsinghua University, 1 Tsinghua Garden Road, Beijing 100084, PR China.
Xu
Lihua
L
Hua
Hui
H
Ren
Chuan
C
Lin
Zhanglin
Z
D001433:Bacteriolysis / Q000235:genetics / Q000528:radiation effects; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects*; D029968:Escherichia coli Proteins / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli