Plasmid vectors, gene cloning

Stoker, N. G., Fairweather, N. F., and Spratt, B. G. (1982). Versatile Low-Copy-Number Plasmid Vectors for Cloning in Escherichia coli. Gene 18 335. 

Gene cloning is a method that uses recombinant technology to insert a gene into a vector DNA (plasmid obtained from a bacterium). The modified vector is put back into the bacterium, which then reproduces endlessly (clones) the new gene as well as the others in the vector. 

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.

Isolation and sequencing of the cellulose synthase gene(s) has not been accomplished yet however, DNA from Acetobacter xylinum containing this gene(s) was cloned into broad host-range plasmid vectors (82). These vectors were mobilized into Pel- mutants to test for complementation. To date, this approach has not produced a pellicle-forming transconjugant from a Pel- mutant of Acetobacter (82). The direct correlation between cellulose production and presence of plasmid DNA in Acetobacter has been reported... 

The BP reaction is a recombination reaction that transfers a DNA fragment (such as a PCR product) flanked by attB recombination sites into an E. coli vector, called Donor vector containing attV sites, to produce a new plasmid, called Entry clone. This reaction is catalyzed by the BP Clonase mix of recombination proteins. In this reaction, the ccdE gene is removed from the Donor vector, thereby allowing the transformants to grow. 

Construction of the gene-bearing vector. Once the clone of DNA has been isolated, this is put into a vector (in this case a plasmid) which will transfer it from the host to the recipient organism. Again, by the use of restriction enzymes to cut the plasmid vector and the cloned DNA, the cloned DNA can now be inserted into the vector. 

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