TY  - JOUR
A1  - Guss, Adam M.
A1  - Rother, Michael
A1  - Zhang, Jun Kai
A1  - Kulkarni, Gargi
A1  - Metcalf, William W.
T1  - New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species
T2  - Archaea
N2  - 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.
KW  - genetics
KW  - site-specific recombination
KW  - tetR
KW  - essential gene
Y1  - 2008
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/7432
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30-73807
SN  - 1472-3654
SN  - 1472-3646
N1  - 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.
VL  - 2
IS  - 3
SP  - 193
EP  - 203
PB  - Heron Publ.
CY  - Victoria, BC
ER  -