New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage PhiC31. Host strains expressing the 
A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage PhiC31. Host strains expressing the PhiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetRgene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.
show moreshow less

Download full text files

Export metadata

  • Export Bibtex
  • Export RIS
Metadaten
Author:Adam M. Guss, Michael Rother, Jun Kai Zhang, Gargi Kulkarni, William W. Metcalf
URN:urn:nbn:de:hebis:30-73807
DOI:http://dx.doi.org/10.1155/2008/534081
ISSN:1472-3654
ISSN:1472-3646
Parent Title (English):Archaea
Publisher:Heron Publ.
Place of publication:Victoria, BC
Document Type:Article
Language:English
Year of Completion:2008
Date of first Publication:2008/10/16
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2010/01/19
Tag:essential gene; genetics; site-specific recombination; tetR
Volume:2
Issue:3
Pagenumber:11
First Page:193
Last Page:203
Note:
Copyright © 2008 Adam M. Guss et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Source:Archaea, 2, S. 193-203 ; doi:10.1155/2008/534081
HeBIS PPN:220829128
Institutes:Biowissenschaften
Dewey Decimal Classification:570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung 3.0

$Rev: 11761 $