Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation...

Golden Gate Assembly is a powerful synthetic biology tool, which utilizes Type IIS enzymes for unidirectional assembly of multiple DNA fragments. The simplicity of its DNA assembly and the exchangeability of standard parts greatly facilitate the generation of combinatorial assembly libraries. Currently there are two popular Golden Gate Assembly frameworks that allow multigene augmentation (MoCl...

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMbeta1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 x 10 transformants per mug of plasmid DNA. For conju...

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. ...

The aim of this work was the construction of a cassette, i.e., a non-replicative molecule formed by linkage of an antibiotic resistance gene and a multiple cloning site. This cassette would allow the cloning and analysis of a wide range of replicons. The aac(6')-lc amikacin gene was isolated and ligated to the multiple cloning site of the pUC18 vector. This construction was HindIII digested and...

objective: the clavulanic acid regulatory gene (clar) is in the clavulanic acid biosynthetic gene cluster that encodes clar. this protein is a putative regulator of the late steps of clavulanic acid biosynthesis. the aim of this research is the molecular cloning of clar, isolated from the iranian strain of streptomyces clavuligerus (s. clavuligerus). materials and methods: in this experimental ...

introduction: follicle stimulating hormone (fsh) is one of the pituitary glycoproteines that it consists of two subunits alpha and beta. the beta subunit is responsible for the biological activity of fsh. the aim of present study was isolation of the β subunit coding sequence containing its signal sequence from human genome and then cloning of the isolated sequence in ppic9 shuttle vector under...

brucellosis is a well-known domestic animal infectious disease, which is caused by brucella bacterium. the outer membrane protein 25 kda (omp25) gene plays an important role in simulating of tnf-α, ifn-α, macrophage, and cytokines cells. in the current study molecular cloning and expression analysis of omp25 gene for designing a subunit vaccine against brucella was investigated. amplifying the ...

The present study was undertaken to clone, express rabies virus glycoprotein (RVG) and to identify potential T-cell epitopes on it. RVG gene (1590 bp) was amplified using gene specific primers. The amplified product was cloned into pTZ57R/T cloning vector by TA cloning. RVG gene was subcloned into pcDNA3.1 (+) expression vector. In this study, cloning and expression of rabies virus glycoprotein...