A Method to Clone and identify the V. stable.

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A Method to Clone and identify the V. stable

Glucuronidase Gene

A thermophilic bacterium, v. stable, has been shown to produce enzyme activity brought about by a thermostable enzyme, Glucuronidase. To develop this enzyme as a new product for a biotechnology company, there are many steps that have to be carried out.

The initial part of the experiment is to isolate the bacterial genome. This is achieved by breaking open the bacterium by treatment with a combination of EDTA and lysozymes. Both of these chemical reagents cause disruption of the cell membrane, hence releasing all of the cells contents to give a cell extract.

This cell extract is then purified by adding a 1:1 mixture of phenol and chloroform to precipitate the protein present, resulting in an aqueous solution of just nucleic acids, i.e. DNA and RNA. However, RNA must be removed and is done so by treatment with the enzyme ribonuclease.

At this stage, the resulting aqueous solution contains just the bacterial DNA, which can then be used in the procedures that follow.

The very first step in the cloning of the glucuronidase gene is to break up the DNA into manageable sized fragments using restriction enzymes. The site of cleavage is at the unique restriction site BAMH1. At the same time the vector is also prepared. The vector chosen for this experiment is pBR322 (see fig below) The reason for using this particular plasmid vector is that it has its own origin of replication, which ensures the multiplication of the vector, it has a copy number that is reasonably high which can be further increased, and two selectable markers, both of which can be involved in insertional inactivation upon fragment insertions.

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The plasmid vector is also exposed to same restriction enzyme as the DNA to linearise it. This is to ensure that the next step is feasible.

Fig: Diagram of pBR322 plasmid:

This next step is to covalently ligate the vector and DNA fragment. For successful ligation, complimentarity of bases is required. This was the purpose of cleaving both DNA and vector plasmid with the same enzyme. The enzyme required for the ligation however, is T4 DNA ligase. The result of this is a circular recombinant molecule.

The recombinant plasmid is now introduced into ...

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