Describe How a Gene Encoding a Therapeutic Protein Could Be Cloned into a Vector to Allow Expression

1626 Words Jun 21st, 2018 7 Pages
A vector is a recombinant DNA carrier, all types have three generic properties; introduction to the host cell can be accomplished easily, each vector contains a replication origin enabling it to reproduce inside the cell and in order to determine which cells contain the vector a simple assay can be carried out, such as, growing the cells on agar plates. At present, there are many different types of vectors available for use the best expression system depends on the gene involved (Hartl, 2011). Examples of vectors include; Plasmids, Bacteriophages, Artificial chromosomes, bacteria, cell free systems and viruses (Klug et al., 2003). The fundamentals of molecular genetics is centred around the “Central Dogma”, this governs protein expression …show more content…
In order to make recombinant DNA the insert must be inserted into the plasmid. To do this, more insert than vector should be added to a new tube and to a ligation kit the following should be added: 2µl of warm vortexed buffer 2, 10µl warm vortexed buffer 1 and 1µl of buffer 3. After this the solution should be incubated at room temp for 15 minutes and then the transformation step should be carried out as below.
Transformation; this is the final step in the subcloning process, it can be described as the incorporation of DNA into a cell. This is done using the “Heat Shock” method in which the volume of the previously ligated plasmid is added to a competent cell and repeatedly incubated on ice or at 47°C or 37°C for variable times after which LB Broth is added and the mixture is shook at 37°C for 1 hour this allows the expression of antibiotic resistance and it has also been found to encourage cell recovery. Then centrifuge the solution and spread on an ampicillin plate and incubate at 37°C overnight to allow the bacteria to grow. This allows the transformation efficiency to be calculated and therefore the number or approximation of cells which took up the antibiotic resistance gene and the required gene to be subcloned (Sara Cormier, 2009).
Subcloning is a technique used in what is known more commonly as gene therapy. It can be defined as the transfer of normal genetic material into a somatic cell that carries one or more mutant alleles, using a vector or
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