Showing papers on "Lambda phage published in 2016"

RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.

Abstract: Wang et al. present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes using combined RecET and Redαβ recombineering systems present within a single E. coli host. Full-length RecE and RecT from Rac prophage mediate highly efficient linear–linear homologous recombination that can be used to clone large DNA regions directly from genomic DNA into expression vectors, bypassing library construction and screening. Homologous recombination mediated by Redαβ from lambda phage has been widely used for recombinant DNA engineering. Here we present a protocol for direct cloning and engineering of biosynthetic gene clusters, large operons or single genes from genomic DNA using one Escherichia coli host that harbors both RecET and Redαβ systems. The pipeline uses standardized cassettes for horizontal gene transfer options, as well as vectors with different replication origins configured to minimize recombineering background through the use of selectively replicating templates or CcdB counterselection. These optimized reagents and protocols facilitate fast acquisition of transgenes from genomic DNA preparations, which are ready for heterologous expression within 1 week.

Orthogonal Modular Gene Repression in Escherichia coli Using Engineered CRISPR/Cas9

Abstract: The progress in development of synthetic gene circuits has been hindered by the limited repertoire of available transcription factors. Recently, it has been greatly expanded using the CRISPR/Cas9 system. However, this system is limited by its imperfect DNA sequence specificity, leading to potential crosstalk with host genome or circuit components. Furthermore, CRISPR/Cas9-mediated gene regulation is context dependent, affecting the modularity of Cas9-based transcription factors. In this paper we address the problems of specificity and modularity by developing a computational approach for selecting Cas9/gRNA transcription factor/promoter pairs that are maximally orthogonal to each other as well as to the host genome and synthetic circuit components. We validate the method by designing and experimentally testing four orthogonal promoter/repressor pairs in the context of a strong promoter PL from phage lambda. We demonstrate that these promoters can be interfaced by constructing double and triple inverter ci...

Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system.

Abstract: Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.

Interaction of Bacterial and Lambda Phage Recombination Systems in the X-Ray Sensitivity of Escherichia coli K-12 (DNA/repair/X bacteriophage red and ganm genes/E. coli recB gene)

Abstract: E. coli cells lysogenic for the thermoin- ducible prophage XcI857 can be transiently induced by a brief heat treatment. Although this treatment does not kill the cells, some X products normally forrned during vegetative phage development are made that can alter the response of host cells to x-irradiation by causing an in- crease in radioresistance. This increased resistance is par- ticularly striking in the recombination-deficient recB- strain, which is normally much more radiosensitive than its recB+ parent. After pulse-heating at 42?, the survival curve of E. coli recB- lysogenized with XcI857 does not differ from that of the wild-type strain. Since X red mutants do not increase the radioresistance of recB strains, both X red gene products, X exonuclease and 3-protein, are re- quired to compensate for the missing recB product. Furthermore, phage-induced radioresistance also occurs in recB+ lysogens even when they carry X red-, but not when the X prophage is gam-. Thus, in wild-type cells, phage-induced radioresistance requires some interaction between the bacterial recB gene product (exonuclease V) and the phage y-protein.