Question

LAB17 Genetic Engineering of Bacteria Problem Is it possible to transfer the allele for resistance to the antibiotic ampicillin into a bacterial cell? Objectives After completing this lab, the student will be able to: 1. Demonstrate micropipetting and sterile pipetting techniques for handling and transferring bacteria and plasmid DNA. 2. Maintain sterile conditions for culturing bacterial cells. 3. Inoculate bacteria into flasks, culture tubes, or agar plates. 4. Culture isolated individual colonies from an agar plate to form genetically identical clones. 5. Determine if antibiotic resistance is present in a bacterial culture. Preliminary Information The bacteria Escherichia coli or E. coli are common bacteria used in microbiology experimentation. These bacteria, found in large numbers in the digestive tracts of mammals, are easily grown on nutrient agar plates. The bacteria have DNA in two areas. There is a single large main loop of DNA and many smaller rings of DNA called plasmids. The plasmids are located in the cytoplasm of the cell, separate from the main DNA ring. In genetic engineering, a piece of DNA from one organism is inserted into another organism. Since the plasma membrane of a cell is very selective about what it allows to cross its membrane and enter the cell, this is not an easy task. One way of getting new foreign DNA into a cell might be to simply inject it. This technique does not work since the cell recognizes the new DNA as foreign and immediately breaks it down. In another technique, the new foreign DNA is combined with host cell DNA that has been removed from the host cell. A specific restriction enzyme is used to cut the foreign DNA from its original chromosome. A restriction enzyme recognizes particular base sequences and cuts at those base sequences. The same restriction enzyme is used to cut open the host plasmid. Using the same restriction enzyme to make both cuts means that the cut ends of the two different DNA pieces will fit together in the next step. Next, the foreign DNA and the cut plasmid and a ligase enzyme are mixed. A ligase enzyme is used to tie the foreign DNA into the plasmid, closing its ring. The new DNA is called recombinant DNA since it is foreign DNA recombined with normal host cell DNA. If this recombinant DNA is then taken into a host cell, it will be recognized as normal cell DNA. The host cell will read the new DNA directions and 153

Answer 1

Yes. The genetically transformed bacteria containing ampicillin resistant gene will survive and grow in the presence of ampicillin.

This is because normally the Escherichia coli or E.coli bacteris does not have resistant gene for any antibiotics including ampicillin. A foreign DNA containing ampicillin resistant gene is transformed into the E.coli plasmid by incorporating restriction and ligase enzymes. Thus the E.coli bacteria with new recombinant DNA will only be able to survive and grow in the presence of ampicillin, other normal bacteria without this ampicillin resistant gene will not survive as they die due to ampicillin toxicity.