Transformation. Making Change Happen

Transcription

1 Transformation Making Change Happen

2 Genetic Engineering Definition: The alteration of an organism s genetic, or hereditary, material to eliminate undesirable characteristics or to produce desirable new ones.

3 Genetic Engineering Techniques Selective Breeding Plants or animals with most desirable characteristics used for further breeding Hybridization (Crossbreeding) Combining different strains of a species to combine the best characteristics of both. (female horse + male donkey=mule) Recombinant DNA Technologies Transformation

4 What is Transformation? The introduction of DNA into a cell or organism, resulting in the expression of a new phenotype. Cornerstone of recombinant DNA technology Has lead to an explosion of new possibilities for health treatments, agricultural applications, environmental solutions

5 How DO we do it? Plasmid DNA + Bacteria =

6 What are Plasmids? Molecules of DNA found in bacteria separate from the bacterial chromosome Small (few thousand base pairs) Usually carry only one or a few genes Circular Can insert gene of interest

7 Plasmids Naturally enter bacteria with ease (protect bacterium from one or more antibiotics). Can be deliberately introduced into bacteria, transforming the cell with incoming genes Replicated by the same machinery that replicates the bacterial chromosome

8 Plasmid we will use: Beta Lactamase (bla) Ampicillin resistance Green Fluorescent Protein (GFP) Aequorea victoria jellyfish gene arac regulator protein Regulates GFP transcription

9 Why use bacteria? Size: Bacteria are unicellular, making them easy to work with. Multicellular organisms are more complex and every cell would need to contain the desired genetic alteration. Reproduction: The faster a model organism reproduces, the more generations of offspring can be quickly produced. Safety: E.coli strain HB101;K-12 does not make people sick.

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13 Fluorescent proteins are a useful tool in biotechnology Provide convenient markers for gene expression and protein targeting in living cells and organisms. Most widely used is green fluorescent protein (GFP) first isolated from the jellyfish Aequorea victoria, can be attached to virtually any protein of interest and still fold into a fluorescent molecule. Can be used to localize previously uncharacterized proteins or to visualize and track known proteins to further understand cellular events. Can be used for process development

14 Aequorea victoria

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16 Recombinant DNA products being used in human therapy Insulin (diabetics) Factor VIII (males w/hemophilia A) Factor IX (hemophilia B) Human growth hormone Erythropoietin (anemia) Angiostatin/endostatin (anti-cancer drugs) leptin

17 The Power of Biotech. Before 1980 s Insulin extracted from the glands of cows and pigs. Worked to lower blood sugar, but seen by the immune system as "foreign" and induced an antibody response, thus reducing the effect and ultimately requiring higher doses. Converted pig insulin into human insulin by removing the one amino acid that distinguishes them and replacing it with the human version. Expensive

18 The Power of Biotech. Insert the human gene for insulin into E. coli and grow recombinant human insulin in culture tanks. E. coli is able to manufacture a fully-functional molecule (trade name = Humulin). Yeast is also used (trade name = Novolin).

19 Methods of Transformation Electroporation Electrical shock makes cell membranes permeable to DNA Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock

20 Basic Procedure Suspend bacterial colonies in Transformation solution (CaCl 2 ) Add pglo plasmid DNA Place tubes in ice Heat-shock at 42 C and place on ice Incubate with nutrient broth Streak plates

21 Purpose of each step: 1. Transformation solution = CaCI 2 Positive charge of Ca ++ ions shields negative charge of DNA phosphates 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression

22 Transformation Procedure See p.33 Student Manual Day 1 Day 2

23 On which plates will colonies grow? Which plates will colonies glow?

24 Corner Stone of Recombinant DNA Technology Completion of Human Genome Project Many human genes have been cloned in E.coli or in yeast. Grow bacteria in bioreactors under conditions that favor synthesis of the material. Can produce unlimited amounts of human proteins in vitro.

25 This material is based upon work supported by the National Science Foundation under Grant No Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.