Name: ______Date: ______Period: _____
Unit 12 Notes, Part 3: Biotechnology B
Ms. Ottolini, AP Biology
A. How can we create recombinant DNA?
1. Let’s say we want to insert a gene from an organism (ex: the human insulin gene) into a bacterial plasmid. The product will be a recombinant plasmid, meaning it contains DNA from two different organisms (a human and a bacterium)
2. If scientists cut the human insulin gene with a restriction enzyme, this will produce “overhangs” of single stranded DNA at the cut site. These “overhangs” are called “sticky ends.” If a bacterial plasmid (a small circle of DNA outside the bacterial chromosome) is cut with the same restriction enzyme, it will have complementary “sticky” ends with the insulin gene and they can be joined using the enzyme DNA ligase.
3. This new plasmid (with DNA from another sample) is considered recombinant DNA because it contains DNA from two different organisms.
4. We can “transform” a bacterial cell by forcing it to “take up” the recombinant plasmid we have created. We do this by placing the bacterial cell in a calcium chloride (CaCl2) solution and “shocking” it with a lot of heat. The combination of these two factors causes holes to form in the bacterial cell’s membrane, through which the plasmid DNA can enter the bacterial cell.
5. When the “transformed” bacterium replicates its plasmid DNA before reproduction by binary fission (cell splitting from one parent cell to two daughter cells), it also replicates the human insulin gene. This is called “gene cloning” and it can produce many copies of a particular gene.
6. When the “transformed” bacterium transcribes and translates its own plasmid DNA, it also transcribes and translates the human insulin gene to produce human insulin protein. This human insulin protein can be “harvested” and used to treat diabetes.
7. Below is a summary diagram showing the process of bacterial transformation and some uses/applications of this technology.
B. How might the AP test assess my knowledge of bacterial transformation?
Two sample scenarios and sets of practice question are given below:
A scientist is using an ampicillinsensitive strain of bacteria that cannot use lactose because it has a nonfunctional gene in the lac operon. She has two plasmids. One contains a functional copy of the affected gene of the lac operon, and the other contains the gene for ampicillin resistance. Using restriction enzymes and DNA ligase, she forms a recombinant plasmid containing both genes. She then adds a high concentration of the plasmid to a tube of the bacteria in a medium for bacterial growth that contains glucose as the only energy source. This tube (+) and a control tube () with similar bacteria but no plasmid are both incubated under the appropriate conditions for growth and plasmid uptake. The scientist then spreads a sample of each bacterial culture (+ and ) on each of the three types of plates indicated to the right.
8. If no new mutations occur, it would be most reasonable to expect bacterial growth on which of the following plates?
(A) 1 and 2 only
(B) 3 and 4 only
(C) 5 and 6 only
(D) 4, 5, and 6 only
(E) 1, 2, 3, and 4 only
9. If the scientist had forgotten to use DNA ligase during the preparation of the recombinant plasmid, bacterial growth would most likely have occurred on which of the following?
(A) 1 and 2 only
(B) 1 and 4 only
(C) 4 and 5 only
(D) 1, 2, and 3 only
(E) 4, 5, and 6 only
In a transformation experiment, a sample of E. coli bacteria was mixed with a plasmid containing the gene for resistance to the antibiotic ampicillin (ampr). Plasmid was not added to a second sample. Samples were plated on nutrient agar plates, some of which were supplemented with the antibiotic ampicillin. The results of E. coli growth are summarized below. The shaded area represents extensive growth of bacteria; dots represent individual colonies of bacteria.
Note: The shaded areas show extensive growth of bacteria and are called “bacterial lawns.” The areas with “dots” show individual colonies of bacteria. The bacteria in these colonies have been successfully “transformed” and are able to live in an environment with ampicillin. Not all bacteria will be successfully transformed, and untransformed bacteria will die, leaving blank spaces surrounding the colonies on the plate.
10. Plates that have only ampicillin-resistant bacteria growing include which of the following?
(A) I only
(B) III only
(C) IV only
(D) I and II
11. Which of the following best explains why there is no growth on plate II?
(A) The initial E. coli culture was not ampicillin-resistant.
(B) The transformation procedure killed the bacteria.
(C) Nutrient agar inhibits E. coli growth.
(D) The bacteria on the plate were transformed.
12. Plates I and III were included in the experimental design in order to
(A) demonstrate that the E. coli cultures were viable
(B) demonstrate that the plasmid can lose its ampr gene
(C) demonstrate that the plasmid is needed for E. coli growth
(D) prepare the E. coli for transformation
C. How can recombinant DNA technology and bacterial transformation be used commercially or medically?
13. As mentioned before, we can cause bacteria to produce the human insulin protein by creating recombinant DNA plasmids containing the human insulin gene. We can use the same method with other human proteins like human growth hormone.
14. Scientists can also use bacteria as “vectors” to introduce foreign genes into other organisms (ex: plants and animals). For example, Agrobacterium tumefaciens is a species of bacteria that infects plant cells and causes the formation of tumors. This condition is called crown gall disease. During infection, a section of a plasmid found within the bacterium (called the Ti plasmid) integrates itself into the plant cell genome. Therefore, scientists can insert a gene from any organism into the Ti plasmid and use the plasmid to deliver the gene to the plant cells. An example of this is golden rice, which has been modified with the gene for beta carotene (Vitamin A) synthesis. This is an extremely beneficial crop in areas of the world where people have vitamin A deficiencies and good conditions for growing rice but not other beta carotene-rich foods (ex: carrots and sweet potatoes).