BACTERIAL CLONING WITH INSULIN BY: B.I.M.P.A INDUSTRIES (BACTERIAL INSERTION AND MANIPULATION OF PLASMIDS IN ANIMALS)

PLASMID INTEGRATION INSTEAD OF HAVING THE BACTERIA INSERT ITS DNA INTO THE THE PLASMID, WE WILL HAVE IT INSERT INSULIN IN ITS PLASMID IN ORDER TO HAVE THE BACTERIA TO PRODUCE INSULIN.

Bacterial plasmid insertion of insulin After many years of hard research, we have found a way to create insulin in the body for people who have diabetes or an insulin deficiency. The methods used in this Recombinant DNA (rDNA) technology are very simple. It involves the insertion of the gene coding for human insulin into a plasmid, which in turn carries the gene into a replicating E. coli bacterium that produces human insulin. Plasmids are small circles of double stranded DNA found in bacteria, separate from the bacterial chromosome and smaller than it. They are able to pass readily from one cell to another, even when the cells are from very different species. Consequently, plasmids can be used as vectors, to carry foreign DNA into a bacterium and replicate/clone it.

How is the insulin gene cloned? 1. RNA that codes for insulin is extracted from pancreatic cells. 2. A complimentary strand of DNA (cDNA) is built onto the RNA using the enzyme reverse transcriptase. 3. Enzymes split the cDNA & RNA strands. 4. The addition of nucleotides & DNA polymerase to the single cDNA strand build a 2nd strand on it.This DNA has special linking sequences built onto each end so it will fit into the plasmid the right way round. 5. The DNA is inserted into a plasmid which also contains a gene for resistance to an antibiotic. To do this a restriction enzyme cuts open the plasmid (the “scissors”), the cDNA is inserted and DNA ligase “glues” the 2 different DNA’s together. This recombinant plasmid now contains a human gene.

How the insulin gene is cloned? 6. The plasmid is mixed in with E. coli & is transfected into the bacteria. 7. Each plasmid replicates, making many copies inside its bacterium (i.e. cloning the gene). 8. The bacteria are put into the antibiotic – only those with the insulin DNA inserted into their plasmid will survive.This is how the transfected bacteria are separated from bacteria without the recombinant DNA in their plasmids. 9. The bacterial culture goes into a fermentation tank where the bacteria (& their plasmids) reproduce. With this recombinant plasmid DNA E. coli can process the instructions to assemble the amino acids for insulin production & they make lots. The trade name for insulin produced from GE E. coli is Humulin. Yeast is also used (trade name = Novolin).

Current Uses Other chemicals manufactured in this way include: Factor VIII for haemophilia A Factor IX for haemophilia B Human growth hormone Erythropoietin- used to boost red blood cells for anaemia Interferons and interleukins for cancer Vaccine antigens AIDS, malaria, and hepatitis

Bioethics, Law and Society Pros: You can have a mass production of insulin to cancel out diabetes. It's easy and efficient It uses your bodies natural bacteria to create insulin It's humane and the technology for it could help cure many other diseases. Cons: We do not know how this will effect all humans. Only a few have been tested and so far they are a success. But we are keeping them under constant supervision because problems may occur over time. If everyone in a population has the same genetic material, a single disease can wipe out the entire population. There might be a malfunction during the cellular cloning that could

Bioethical Considerations Why or Why Not? What about all the animal testing that have been, you might ask? The animals in the testing are well taken cared of and fed well and sheltered. We make sure to treat them with respect and take very good care of them. Any abusive act or violence towards our test animals will not tolerated. When they are no longer needed they are disposed of in a nice clean safe way, so that they don't contaminate the environment. It's inexpensive and can/may cure HIV's permanently and other related diseases.

Case studies We have used the E-Coli bacterium from animals(mostly pigs because they are very compatible with humans), and have been able to insert the insulin gene DNA into its plasmid. Therefore, the bacterium will then start to produce insulin that we take purify and inject into people who have diabetes. This has worked very well, but now we are are able to do it human, and literally cure diabetes. It also works for other diseases like cancer, hemophilia, AIDS, etc. With this technology we will be able to eradicate some of the most heinous diseases.

References AASwdPC Textbook PowerPoint notes ng+Insulin+Gene.doc