Abstract
A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro.
Highlights
Shewanella oneidensis MR-1, a facultatively anaerobic c-proteobacterium, is capable of utilizing a diversity of organic compounds and metals to obtain energy needed for growth and survival
Since the genome sequence of S. oneidensis MR-1 was released in 2002 [1], many biological processes in S. oneidensis MR-1 have been investigated in a high-throughput manner by utilizing methods such as whole-genome microarrays, liquid chromatography-mass spectrometry (LC/MS), gas-chromatography-mass spectrometry (GC/MS), and nuclear magnetic resonance (NMR)
Such site-specific recombination based cloning systems have been successfully employed for building a number of clone sets [18,20,21,22,40,41,42]
Summary
Shewanella oneidensis MR-1, a facultatively anaerobic c-proteobacterium, is capable of utilizing a diversity of organic compounds and metals to obtain energy needed for growth and survival. A collection of such clones should be flexible and amenable for many in vivo and in vitro experimental systems as discussed in recent reports and reviews [18,19,20,21,22] This resource would facilitate research in various aspects of functional genomics and proteomics in S. oneidensis, including but not limited to deciphering the functional activities of proteins, especially those involved in metal reduction [23,24,25], and building regulatory networks and interaction maps [26,27]
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