共查询到20条相似文献,搜索用时 187 毫秒
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Pricila Hauk Kristina Stephens Ryan Mckay Chelsea Ryan Virgile Hana Ueda Marc Ostermeier Kyoung-Seok Ryu Herman O. Sintim William E. Bentley 《Nucleic acids research》2016,44(21):10515-10525
Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering. 相似文献
13.
Adam M. Guss Michael Rother Jun Kai Zhang Gargi Kulkkarni William W. Metcalf 《Archaea (Vancouver, B.C.)》2008,2(3):193-203
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 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 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 approximately one half or one fourth as active, respectively.
The CTG fusion was 95% less active than the ATG fusion. 相似文献
14.
BioLogic gates enable logical transcription control in mammalian cells 总被引:11,自引:0,他引:11
15.
16.
17.
18.