A Method to Clone and identify the V stable
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.
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 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. If the fragment insert has occurred at the BAMH1 site in the plasmid then the tetracycline resistance gene is inactivated. Therefore the first thing to do is to grow all host cells, transformants or not, onto an agar plate containing ampicillin. Then with a sterile toothpick, transfer a small amount of each colony to an identified spot on an agar containing tetracycline. Those colonies that fail to grow have been transformed with a piece of the original bacterial DNA.
The product of the experiment at this stage is called a gene bank or library.
To obtain the colony that carries the fragment with the gene encoding glucuronidase, further selective testing is required. This process is screening. A specific example of screening that would be ideal for this particular experiment is direct selection. This is because the phenotype of the recombinant can be easily differentiated from that of non-recombinants.
If the gene expresses glucuronidase, it cleaves its substrate, X-gluc, to give a distinct colourless to bright blue change.
Therefore, in this case, all the recombinants of different fragments can be plated onto an agar containing the minimal medium, X-gluc. Those colonies that undergo this colour change are those that suggest the presence of the gene on that particular fragment. 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.