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Title: 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 φC31. Host strains expressing the φC31 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 P mcrB 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 tetR gene. 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 approximatelymore » one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less
Authors:
 [1] ;  [2] ;  [3] ;  [3] ;  [3]
  1. Department of Microbiology, University of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Goodwin, Urbana, IL 61801, USA, Departments of Organismic and Evolutionary Biology, and Microbiology and Molecular Genetics, Harvard University, 16 Divinity Ave, Biolabs 4081, Cambridge, MA 02143, USA
  2. Department of Microbiology, University of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Goodwin, Urbana, IL 61801, USA, Institut für Molekulare Biowissenschaften, Molekulare Mikrobiologie und Bioenergetik, Johann Wolfgang Goethe-Universität, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
  3. Department of Microbiology, University of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Goodwin, Urbana, IL 61801, USA
Publication Date:
OSTI Identifier:
1198220
Grant/Contract Number:
FG02-02ER15296
Type:
Published Article
Journal Name:
Archaea
Additional Journal Information:
Journal Volume: 2; Journal Issue: 3; Related Information: CHORUS Timestamp: 2016-08-23 06:22:05; Journal ID: ISSN 1472-3646
Publisher:
Hindawi Publishing Corporation
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English