Example of (he Use of Plasmid Cloning, pBR32Z

Example of (he Use of Plasmid pBR32Z as a Vecior: Expression In E. coil of a Chemicaly synthesized Gene for the Hormone Somalostatin
There are iwo reasons rot selecting this particular experiment to illustrate the use of pBR322 as a vector. Firstly, the [act that gene manIpulation has bccn successfully uscd to obtain a functional gene product from a chemically synthesized gene indicates that the potential of genetic engineering is not just a dream but has already been realized. Secondly, numerous elegant ‘tricks’ were used to ensure success and they warrant detailed examination for they iUustrate the versatility of the basic techniques of gene manipulation.
The rationale of the experiment was in fact to show that recombinant DNA technology can be used to fuse chemically synthesized genes to plasmid clcmcnts for expression in E. coil or other bacteria. As a model, kakura ti ul. (1977) designed and synthesized a gene for the small hormone somatostatin. The somatostatin ‘gene’ was chosen because sonsatostatin is a small polypcplidc of known nmino acid coaiposition, hcrc arc scnsitivc radio-immune and biological assays, and, being a hormone it is of intrinsic biological interest. The methods used to chemically synthesize the somatostatin gene are outside the scope of this discussion. Suffice it to say that it was constructed with an Eco RI site at one end and a Barn HI site at the other (Fig. 3.5).
Also, a methionine codon Precedes the normal NHrterminal amino.acid of somalostarin and the COOH-erminal is followed by two nonsense codons.Example of (he Use of Plnsmid pBR32Z as a Vecior: Expression In E. coil of a ChemIcall SaSheslzed Gene toe the Hormone Somalostatin There are iwo reasons rot selecting this particular experiment to illustrate the use of pBR322 as a vector. Firstly, the [act that gene manIpulation has bccn successfully uscd to obtain a functional gene product from a chemically synthesized gene indicates that the potential of genetic engineering is not just a dream but has already been realized. Secondly, numerous elegant ‘tricks’ were used to ensure success and they warrant detailed examination
for they iUustrate the versatility of the basic techniques of gene manipulation. The rationale of the experiment was in fact to show that recombinant DNA technology can be used to fuse chemically synthesized genes to plasmid clcmcnts for expression in E. coil or other bacteria. As a model, kakura ti ul. (1977) designed and synthesized a gene for the small hormone somatostatin. The somatostatin ‘gene’ was chosen because sonsatostatin is a small polypcplidc of known nmino acid coaiposition, hcrc arc scnsitivc radio-immune and biological assays, and, being a hormone it is of intrinsic biological interest. The methods used to chemically synthesize the somatostatin gene are outside the scope of this discussion. Suffice it to say that it was constructed with an Eco RI site at one end and a Barn HI site at the other (Fig. 3.5). Also, a methionine codon Precedes the normal NHrterminal amino.acid of somalostarin and the COOH-erminal is followed by two nonsense codons.

In the first part of Ihe ezperiment ihree new pasmids were cteaied from pBR322. The control region ol the lx opcron. comprising the lx promOter, cRLaboIIte gcne activalor-prOtán binding site, the opcr.tor, the rbosomc binding site, and the first seven triplets of the -gaL%ctosidasc strucural

gene. re imened into the Ecu RI sate or pBR322 to create p1W 10. P1ainkI pBHIO has two Leo RI sites and onc of these was rcrnocd to generate p111420. Finally, the synthetic sornlltost4tic gene wa insrtcd nct to the Sac conuol gene to yield pSom I.