Lesson 1 Introduction to Genetic Engineering

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1 Lesson 1 Introduction to Genetic Engineering Genetic engineering is the manipulation of an organism s genetic material (DNA) by introducing or eliminating specific genes A gene is a piece of DNA which mostly provides the instructions for making (codes for) a protein This protein gives an organism a particular trait DNA RNA PROTEIN TRAIT

2 Fig. 20-2a Bacterium 1 Gene inserted into plasmid (vector) Recombinant DNA (plasmid) Cell containing gene of interest Bacterial chromosome Plasmid 2 2 Gene of interest Plasmid put into bacterial cell DNA of chromosome Recombinant bacterium

3 (1) Recombinant DNA Technology Recombinant DNA technology is the process of cutting and recombining DNA fragments as a means to isolate genes or to alter their structure and function Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Cloned genes are useful for making copies of a particular gene and producing a protein product

4 Plasmids Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes

5 (2)Genetic transformation Genetic transformation involves the transfer of a gene which commonly have been cloned into a vector such as a plasmid into an organism in order to change the organism s trait In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid

6 Conceptual points Transformation Solution : It is postulated that the Ca 2+ cation of the transformation solution (50 mm CaCl 2, ph 6.1) neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells. Heat Shock :The heat shock increases the permeability of the cell membrane to DNA. While the mechanism is not known, the duration of the heat shock is critical and has been optimized for the type of bacteria used and the transformation conditions employed.

7 Applications of genetic transformation Agriculture Resistance of frost, pest, spoilage Bioremediation Digest oil spill Medicine Protein drugs, Subunit vaccine, Gene therapy

8 Fig. 20-2b Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the cloned gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Protein harvested Basic research on gene 4 Basic research and various applications Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth

9 pglo Gene Regulation encodes the gene for Green Fluorescent Protein (GFP) encodes a gene (bla) for resistance to the antibiotic ampicillin also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells

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11 lac operon-- an inducible operon An inducible operon is one that is usually off by binding a active repressor For example, the lac operon of E. coli The lac operon contains genes that code for enzymes used in the hydrolysis and metabolism of lactose

12 Fig. 18-4a Regulatory gene Promoter Operator DNA laci lacz mrna 5 3 RNA polymerase No RNA made Protein Active repressor (a) Lactose absent, repressor active, operon off

13 Fig. 18-4b lac operon DNA laci lacz lacy laca mrna 5 3 RNA polymerase mrna 5 Protein b-galactosidase Permease Transacetylase Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on An inducer can inactivate a repressor to turn an inducible operon on

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16 Lesson 2 Transformation Procedure (containing 50mM CaCl 2, ph6.1)

17 0.8mg/10ml (10 min)

18 (2 min)

19 (10 min)

20

21 (37 over night)

22 Lesson 3 Data Collection and Analysis

23 Lesson 4 Calculate Transformation Efficiency

24 Questions 1. On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? Explain your prediction. 2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your prediction. 3. Which plates should be compared to determine if any genetic transformation has occurred? Why? 4. How do you interpret the difference if you compared the (-) pglo LB/amp plate with the (-) pglo/lb plate. 5. How do you interpret the difference if you compared the (+) pglo LB/amp plate with the (+) pglo LB/amp/ara plate

25 6. From the results that you obtained, how could you prove that these changes that occurred were due to the procedure that you performed? 7. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful. 8. Very often an organism s traits are caused by a combination of its genes and the environment it lives in. Think about the green color you saw in the genetically transformed bacteria:

26 a. What two factors must be present in the bacteria s environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you look at the bacteria). b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria turn green? c. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

27 Report 封面 實驗名稱 : Transformation 組別 : 組員 : 實驗目的與原理 (Lesson 1, Lesson 2) 材料與儀器 (Lesson 2) 實驗步驟 (Lesson 2) 結果與討論 (Lesson 3, Lesson 4) 回答問題