scholarly journals A highly efficient targeted recombination system for engineering linear chromosomes of industrial bacteria Streptomyces

2018 ◽  
Vol 64 (4) ◽  
pp. 167-173 ◽  
Author(s):  
Hung-Yin Pan ◽  
Carton W. Chen ◽  
Chih-Hung Huang
Keyword(s):  
Highly Efficient ◽  
Recombination System ◽  
Linear Chromosomes ◽  
Targeted Recombination

scholarly journals Tn502 and Tn512 Are res Site Hunters That Provide Evidence of Resolvase-Independent Transposition to Random Sites

2010 ◽  
Vol 192 (7) ◽  
pp. 1865-1874 ◽  
Author(s):  
Steve Petrovski ◽  
Vilma A. Stanisich
Keyword(s):  
Alternative Pathway ◽  
Target Selection ◽  
Target Molecule ◽  
Second Stage ◽  
Highly Efficient ◽  
Recombination System ◽  
Predominant Product ◽  
Random Target

ABSTRACT In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.

scholarly journals Switching Species Tropism: an Effective Way To Manipulate the Feline Coronavirus Genome

2003 ◽  
Vol 77 (8) ◽  
pp. 4528-4538 ◽  
Author(s):  
Bert Jan Haijema ◽  
Haukeliene Volders ◽  
Peter J. M. Rottier
Keyword(s):  
Recombinant Virus ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Rna Recombination ◽  
Recombination System ◽  
Lethal Infection ◽  
Positive Strand Rna ◽  
Targeted Recombination ◽  
Selection Of

ABSTRACT Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPVΔ7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPVΔ7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.

scholarly journals New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes forMethanosarcinaspecies

Archaea ◽  
2008 ◽  
Vol 2 (3) ◽  
pp. 193-203 ◽  
Author(s):  
Adam M. Guss ◽  
Michael Rother ◽  
Jun Kai Zhang ◽  
Gargi Kulkkarni ◽  
William W. Metcalf
Keyword(s):  
Chromosomal Integration ◽  
Recombination Site ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Highly Efficient ◽  
Recombination System ◽  
Regulated Gene Expression ◽  
Hybrid Promoters ◽  
Site Specific Recombination System

A highly efficient method for chromosomal integration of cloned DNA intoMethanosarcina spp.was developed utilizing the site-specific recombination system from theStreptomycesphage 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 expressedM. barkeriPmcrBpromoter 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 thetetRgene. 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 withtetR-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.

scholarly journals Contribution of Pentose Catabolism to Molecular Hydrogen Formation by Targeted Disruption of Arabinose Isomerase (araA) in the Hyperthermophilic Bacterium Thermotoga maritima

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Derrick White ◽  
Raghuveer Singh ◽  
Deepak Rudrappa ◽  
Jackie Mateo ◽  
Levi Kramer ◽  
...  
Keyword(s):  
Molecular Hydrogen ◽  
Thermotoga Maritima ◽  
Mutant Cell ◽  
Complex Medium ◽  
Content Type ◽  
Genetic Method ◽  
Hyperthermophilic Bacterium ◽  
Recombination System ◽  
Targeted Recombination ◽  
Arabinose Isomerase

ABSTRACT Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H2). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H2 yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H2 using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H2 production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H2 formation and life at an extremely high temperature in the bacterial domain. IMPORTANCE We describe here the development of a genetic system for manipulation of Thermotoga maritima. T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H2) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H2 yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H2 formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales.

Highly efficient and selective photocatalytic CO2 to CO conversion in aqueous solution

2020 ◽  
Vol 56 (27) ◽  
pp. 3851-3854 ◽  
Author(s):  
Xiaomin Chai ◽  
Hai-Hua Huang ◽  
Huiping Liu ◽  
Zhuofeng Ke ◽  
Wen-Wen Yong ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Photocatalytic Performance ◽  
Aqueous Media ◽  
Highly Efficient ◽  
Co Conversion

A Co-based complex displayed the highest photocatalytic performance for CO2 to CO conversion in aqueous media.

Tuning the pore structures and photocatalytic properties of a 2D covalent organic framework with multi-branched photoactive moieties

Nanoscale ◽  
2020 ◽  
Vol 12 (30) ◽  
pp. 16136-16142
Author(s):  
Xuan Wang ◽  
Ming-Jie Dong ◽  
Chuan-De Wu
Keyword(s):  
Photocatalytic Properties ◽  
Effective Strategy ◽  
Covalent Organic Framework ◽  
Highly Efficient ◽  
Pore Structures ◽  
Local Connection ◽  
Organic Framework

An effective strategy to incorporate accessible metalloporphyrin photoactive sites into 2D COFs by establishing a 3D local connection for highly efficient photocatalysis was developed.

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