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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. ...read more.


The recombinant plasmid is now introduced into a suitable host cell, in this case E-coli. This is done by the method of transformation, where competent cells are prepared via treatment with calcium chloride. This makes them bind DNA on their cell walls. For the cells to uptake the bound DNA, it is exposed to a short pulse of elevated temperature, which partially fluidises the membrane. However the frequency of transformation obtained in this way is not high, therefore another method called electroporation can be used. This utilises exposure of competent cells to a high voltage pulse and gives transformation frequencies 100 times higher than those obtained by chemical methods. The host cells that have taken up plasmid are termed transformants and those that have taken up plasmid containing foreign DNA are called recombinants. Not all host cells can be termed transformants, as some may not have taken up the plasmids. Therefore the transformants have to be selected. The plasmid vector pBR322 has two antibiotic resistance genes, one for ampicillin and the other for tetracycline. ...read more.


White colonies, however, are those recombinants that do not contain this particular gene. At this point, the gene that encodes glucuronidase has been cloned and identified from the thermophilic bacterium, V.stable. If my bosses were to decide that a recently discovered exotic fungus containing a more interesting glucuronidase activity, I would modify my approach by changing the vector into which the fungal DNA is inserted and the host into which the recombinant vector is introduced. The vector I would propose to use would be a yeast expression vector, YAC, or Yeast Artificial Chromosomes because they allow large pieces of DNA (average 600 kb) to be maintained in yeast. It also means that a good coverage of a large genome in a small number of clones can be achieved. This vector still contains the required components to enable the glucuronidase gene to be selected for (i.e. a selectable marker). The YACs are already linear, therefore a step of the original method is reduced. Because of this, the ideal host cell would be a yeast cell. Overall, the above process will enable a variety of different organisms that contain the glucuronidase gene to be successfully cloned and identified efficiently. ...read more.

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