Recombinant plasmid vectors

In both methods, cultured animal cells must be treated to facilitate their initial uptake of a recombinant plasmid vector. This can be done by exposing cells to a preparation of lipids that penetrate the plasma membrane, Increasing Its permeability to DNA. Alternatively, subjecting cells to a brief electric shock of several thousand volts, a technique known as electroporation, makes them transiently perme able to DNA. Usually the plasmid DNA Is added In sufficient concentration to ensure that a large proportion of the cultured cells will receive at least one copy of the plasmid... 

Plasmid Vectors for Facile Introduction of Passenger DNA and Selection of Recombinants. The map of a commonly used plasmid vector, pUC19 (7), is shown in Figure 2. Three parts of the vector are key to its utility. The origin sequence, oh, allows the repHcation of plasmid DNA in high copy number relative to the chromosome. A gene, amp, encoding the enzyme beta-lactamase, which hydrolyzes penicillin compounds, allows... 

E. coli strain BL21(DE3) (F ompT ksdSafra ms ) gal dcm) (Novagen, Madison, WI, USA) was used as a host for recombinant OPH expression. Recombinant plasmids pTOH and pEOH that contain OPH gene fused with hexa-histidine affinity tag under trc and T7 promoter, respectively, as control vectors and pTTOH and pETOH that contain OPH gene fused with Tat signal sequence and hexa-histidine affinity tag under trc and T7 promoter, respectively, were used (Fig. 1). 

Fig. 1 Schematic outline of procedures employed in the synthesis of a cDNA gene copy from a polyadenylated mRNA template, insertion of the cDNA into a bacterial plasmid vector by a homopolymer tailing strategy, and cloning of the recombinant plasmid in an Escherichia coli host.

One usually tries to adjust experimental conditions so that only one plasmid or phage is introduced into a single cell. When that cell replicates on a plate of nutrient agar, it will produce millions of cells until a colony becomes visible to the naked eye. If cells are sufficiently diluted before they are spread onto the plate, each colony will consist of a clone derived from a single ancestral cell. If each cell originally contained only one copy of the phage or plasmid vector, each colony will contain recombinant DNA that is homogeneous. That is, each colony will contain cloned DNA. 

Most of the known microorganisms to be active for BDS (by the date that patent [408] was introduced), were considered in this invention. Furthermore, their mutational or engineered derivatives, enzymes, cell-free extracts, recombinant enzymes, recombinant DNA, plasmids, vectors, and fragments were also incorporated in the intellectual property document. The mentioned operating conditions regard ambient temperature, mechanical agitation and a 1 9 biocatalyst/petroleum ratio. 

Plasmids. See also Plasmid vectors expression, 12 474 mobilizable, 12 471 in salicylic acid manufacture, 22 8 Plasmid vectors, for passenger DNA introduction and recombinant selection, 12 501 Plasmin, 5 175... 

Recombinant selection, 12 517 plasmid vectors for, 12 501 Recombinant technology, 11 11-14 Recombinant therapeutics... 

Once the recombinant vectors have been produced, they are used to transform host cells. In the example of the plasmid pBR322, the host cells are bacteria. Once transformed, the bacteria are plated on selective media so that bacteria transformed with a recombinant plasmid can be easily identified. In the case of plasmid pBR322 shown in Figure 1-6-3, bacteria with recombinant plasmids would be resistant to ampicillin but sensitive to tetracycline. 

Chemical recombination of vector insertion into the host cell recombining plasmid genes into the host genetic complement Replication of host cell... 

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