NDLI: Gene Replacement in Mycobacteria by Using Incompatible Plasmids

Content Provider	PubMed Central
Author	Pashley, Carey A. Parish, Tanya Mcadam, Ruth A. Duncan, Ken Stoker, Neil G.
Copyright Year	2003
Abstract	A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.
Related Links	http://dx.doi.org/10.1128/aem.69.1.517-523.2003
Ending Page	523
Page Count	7
Starting Page	517
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	1
Volume Number	69
Language	English
Publisher	American Society for Microbiology
Publisher Date	2003-01-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes

Transformation of Rhizobia with Broad-Host-Range Plasmids by Using a Freeze-Thaw Method

Transformation of the Antibacterial Agent Norfloxacin by Environmental Mycobacteria

Predominance of a Versatile-Peroxidase-Encoding Gene, mnp4, as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus

First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening▿ †

Structural Analysis of Biofilm Formation by Rapidly and Slowly Growing Nontuberculous Mycobacteria▿

Uptake of Carbon Monoxide and Hydrogen at Environmentally Relevant Concentrations by Mycobacteria†

Quantitative Analysis of Bacterial Gene Expression by Using the gusA Reporter Gene System

Gene Replacement in Mycobacteria by Using Incompatible Plasmids

Similar Documents

Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

An Efficient Method of Selectable Marker Gene Excision by Xer Recombination for Gene Replacement in Bacterial Chromosomes

Transformation of Rhizobia with Broad-Host-Range Plasmids by Using a Freeze-Thaw Method

Transformation of the Antibacterial Agent Norfloxacin by Environmental Mycobacteria

Predominance of a Versatile-Peroxidase-Encoding Gene, mnp4, as Demonstrated by Gene Replacement via a Gene Targeting System for Pleurotus ostreatus

First Detection of Mycobacteria in African Rodents and Insectivores, Using Stratified Pool Screening▿ †

Structural Analysis of Biofilm Formation by Rapidly and Slowly Growing Nontuberculous Mycobacteria▿

Uptake of Carbon Monoxide and Hydrogen at Environmentally Relevant Concentrations by Mycobacteria†

Quantitative Analysis of Bacterial Gene Expression by Using the gusA Reporter Gene System

Gene Replacement in Mycobacteria by Using Incompatible Plasmids