TY - JOUR
T1 - High-throughput screening of bacterial protein localization.
AU - Werner, John N.
AU - Gitai, Zemer
N1 - Funding Information:
The authors thank Eric Chen for his assistance in creating the Caulobacter ORFeome and imaging, Jonathan Guberman for writing the image acquisition and image scoring programs, Angela Zippilli and Joe Irgon for gene sequencing and validation, and the entire Gitai lab for assistance with image scoring. We also thank Greg Phillips (Iowa State University, Ames, IA), Michael Kahn (Washington State University, Pullman, WA), Martin Thanbichler (Max Planck Institute for Terrestrial Microbiology, Marburg, Germany), and M. R. Alley (Anacor Pharmaceuticals, Palo Alto, CA) for materials, and Denis Dupuy and the rest of Marc Vidal's lab for help in constructing the Caulobacter ORFeome entry library. John N. Werner is supported by a postdoctoral fellowship, Grant 1F32AI073043-01A1, from the National Institute of Allergy and Infectious Diseases and Zemer Gitai is supported by funding from Grant DE-FG02-05ER64136 from the U.S. Department of Energy Office of Science (Biological and Environmental Research) and a National Institute of Health New Innovator Award Number 1DP2OD004389-01.
PY - 2010
Y1 - 2010
N2 - The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where each specific protein is subcellularly localized. We have developed a pipeline of methods that allows rapid, efficient, and scalable gene cloning, imaging, and image analysis. This work focuses on a high-throughput screen of the Caulobacter crescentus proteome to identify proteins with unique subcellular localization patterns. The cloning, imaging, and image analysis techniques described here are applicable to any organism of interest.
AB - The ever-increasing number of sequenced genomes and subsequent sequence-based analysis has provided tremendous insight into cellular processes; however, the ability to experimentally manipulate this genomic information in the laboratory requires the development of new high-throughput methods. To translate this genomic information into information on protein function, molecular and cell biological techniques are required. One strategy to gain insight into protein function is to observe where each specific protein is subcellularly localized. We have developed a pipeline of methods that allows rapid, efficient, and scalable gene cloning, imaging, and image analysis. This work focuses on a high-throughput screen of the Caulobacter crescentus proteome to identify proteins with unique subcellular localization patterns. The cloning, imaging, and image analysis techniques described here are applicable to any organism of interest.
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U2 - 10.1016/s0076-6879(10)71011-9
DO - 10.1016/s0076-6879(10)71011-9
M3 - Article
C2 - 20946849
AN - SCOPUS:79952196574
SN - 0076-6879
VL - 471
SP - 185
EP - 204
JO - Methods in enzymology
JF - Methods in enzymology
ER -