In order to access the gene that is responsible for the production of insulin in humans, the DNA it is found on, must be extracted from the human cell. This is primarily done by breaking the cell membrane and then the nuclear membrane of the human cell, allowing having direct access to the chromosomes which will actually lead onto identifying the exact location (Gene Mapping) of the human insulin gene on the chromosome.
The next step is to isolate the gene, and this is done by using restriction enzymes, as they recognise and cut the DNA sequence which codes specifically for the insulin. The restriction enzyme used, is the one derived from E.coli bacteria-Eco RI- as this type of bacteria is used as the hosts for the insulin hormone production. Eco RI recognises the sequence “GAATTC” on the forward strand and “CTTAAG” on the parallel strand of the DNA, forming staggered cuts between the G (Guanine) and the A (Adenine) on both strands.
Using PCR (Polymerase Chain Reaction), the isolated human insulin gene is copied in order to allow a very large number of genes available to work with. For confirming the sizes (how many base pairs long the gene sequence is) of the insulin genes that were copied, a gel is run as Gel Electrophoresis is performed. The DNA ladder being used identifies the size of the insulin gene being 1430 base pairs long. (Genetic Home Reference, 2015)
These staggered cuts leave sticky ends, which can form hydrogen bonds with the complementary sequence on the E.coli plasmids (circular DNA found in bacteria) which are also cut using the Eco RI restriction enzyme once it is removed from the bacteria cell. The cutting process of the restriction enzyme Eco RI opens up the plasmid ring. The whole principle behind this is that when the E.coli plasmids and the Human insulin gene is cut using the same restriction enzyme being -Eco RI- both the DNA molecules produce fragments that have the same complementary sticky ends, therefore this makes it possible for the sticky ends from the bacterial plasmid bonding with the sticky ends of the Human insulin gene when the two are brought together. Forming the phosphordiester bonds to seal the sugar-phosphate backbones is completed with the addition of the DNA ligase enzyme, as it creates the bonds.
The recombinant bacterial DNA, containing the human insulin gene, is reinserted into the E.coli bacteria cell. To accomplish the insertion of the plasmids, the bacteria cells must reach a state of competency; this is done by giving het shocks and colds shocks along with the CaCl2. Only when these are performed the permeability and the structure of the cell wall and membrane change, allowing the newly combined plasmids to be absorbed into the bacteria cell. This step is called, Bacterial Transformation.
Nutrient is given to the bacteria cell to maintain its function; to divide and live. Meanwhile they produce the human insulin, as the bacterial cells function and activate the human insulin gene. Therefore, as the bacteria cells grow and divide, the human insulin gene is passed down to the newly formed cells, allowing the insulin to produce furthermore.
With the fermentation process, large quantities of bacteria are produced, and when it is reached to a sufficient amount, the bacteria containing the recombinant DNA are removed from the fermentation tanks in order to gather and purify the human insulin protein molecules the bacteria produce. The bacteria cells are destroyed once the process comes to an end.
Nowadays, people with diabetes acquire human insulin from bacterial sources which is advantageously compatible with their own bodies, not at all different from the insulin produced naturally by humans. (778 words)
by
zeynep_merve10hotmailcouk (student)
