1. Biotechnology Part 2 Insulin made by E.coli. The last lecture we were talking about how to cut a gene out of the host DNA and transfer it into a bacterial cell. We ended with Gel Electrophoresis, to isolate the desired gene, and PCR, to make many copies of the gene.

2. 3. The plasmid vector is cleaved by same restriction enzymes that were used to excise the insulin gene. –The reason that the same enzymes are used on both is because they make identical sticky ends so gene will fit and bind into plasmid. Imagine that you are cutting a strip of paper with patterned scissors. If you were to cut one strip with one pattern and another strip with a different pair of patterned scissors, the two strips of paper would not match up with each other. Restriction enzymes are essentially doing the same thing. –The cut plasmid and the insulin gene are placed into a test tube with each other and enzyme, ligase, is added. The ligase is like a needle and thread. It is responsible for binding the plasmid and gene sticky ends together.

3. 4. When the gene has been successfully inserted into the plasmid vector, the vector is then transformed into the target cell. –Target cells are made competent, chemically or electrically, by removing the cell wall. –Competent cells (with the cell wall removed) are called protoplasts. –The vector is then added to a test tube full of competent cells. –The competent cells can then take up or accept the extracellular DNA. –Next, the cells are grown in nutrient broth to promote healing and growth for a short time before they are streaked on a plate for colony separation. Notice that on the plasmid map in Fig. 10.8, there were a couple of antibiotic resistance genes. These genes are called reporter genes. –Reporter genes help researchers know if the vector made it into the cell quickly without having to isolate the plasmid.

4. On the plasmid map one the genes is for ampicillin resistance. So when the nutrient plates are made for colony isolation of the genetically engineered cells, some ampicillin is added to the media. The ampicillin will kill any cell that does not contain the plasmid for ampicillin resistance. –The ampicillin gene is the reporter gene because when a cell survives and a colony is grown, we know that the plasmid we inserted is present in the cell because it is alive. –Any other cells that don’t have the plasmid die because of the ampicillin.

5. Once a colony is identified with the engineered plasmid, it is then grown in large quantities. Then the protein product, in our case insulin, is harvested.

6. Uses of genetically modified organisms Microorganisms are used for making proteins and enzymes and hormones because they grow quickly and are easily manipulated. –Not only that, but no one complains if thousands of bacteria are killed for the sake of research or genetic engineering. In addition to the proteins they make, sometimes the genetically modified organisms themselves are used. –Ie. Bioremediation is the use of organisms that can break down toxic chemicals. This is a great way to clean up toxic spills. –In addition, new microbes are engineered for pest control for crops.

7. However, here is something to think about. –What happens when we introduce a new microbe into the environment that never existed before? –How will its presence change the balance of the ecosystem? –Could we inadvertently create an organism that might cause more harm to the environment? –On the other hand, so much good has and can be done, should those endeavors stop simply because we don’t know what the long term effects (good or bad) will be?

8. Subunit vaccines are another example of the importance of genetic engineering. –The gene that causes disease for a particular bacterial organism is placed in the vaccine. –Advantage: no chance of becoming infected with the disease because there is no organism present in the vaccine. –Disadvantage: time consuming, expensive, and DNA is quickly degraded in the body.

9. The homework for biotechnology is now on the web. It will be due Friday, Oct. 13, 2006 by midnight.