GENE CLONING AND RECOMBINANT DNA TECHNOLOGY

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1 GENE CLONING AND RECOMBINANT DNA TECHNOLOGY

2 What is recombinant DNA? DNA from 2 different sources (often from 2 different species) are combined together in vitro. Recombinant DNA forms the basis of cloning. Why is this useful? Allows scientists to manipulate organisms (e.g. E. coli) to make useful products known as recombinant proteins. Can be used to modify the phenotype of an organism. These are called genetically modified organisms (GMO). Used in biotechnology, medicine and research.

3 Generating recombinant DNA HOW TO CLONE A GENE!

4 THE PRINCIPE OF CLONING

5 The cloning toolkit

6 Step 1 in gene cloning Vector DNA acts as a carrier for the DNA segment to be cloned When a vector is introduced into a living cell, it can replicate making many copies Common vectors are plasmid or viral Also need the gene of interest from chromosomal DNA

7 Plasmid DNA Plasmids are found in bacterial cells. Small circular DNA molecules that replicate separately from the bacterial chromosome.

8 Plasmids as a tool for cloning Origin of replication Gene encoding a selectable marker Promoter Multiple cloning site

9 A typical bacterial cloning vector Antibiotic resistance gene Multiple cloning site Bacterial promoter Origin of replication

10 Preparing the DNA for cloning

11 Restriction enzymes and cloning Enzymes that cut the DNA at a limited number of specific locations Discovered in the late 1960s by biologists doing basic research on bacteria In bacterial these protect the cell by cutting up DNA from other organisms such as phages Hundreds of different restriction enzymes have been identified Each enzyme recognises and cuts at a very specific sequence of DNA

12 RESTRICTION ENZYMES AND CLONING

13 RESTRICTION ENZYMES AND CLONING

14 RESTRICTION ENZYMES AND CLONING

15 Inserting your gene of interest into the cloning vector

16 Ligating your Gene of Interest into the Cloning Vector

17 DNA LIGASE

18 Making complementary DNA (cdna) from eukaryotic genes

19 BACTERIAL TRANSFORMATION

20 Bacterial transformation Goal for recombinant vector to be taken up by bacteria Some will take up a single plasmid Most cells fail to take up a plasmid Vector carries a selectable marker Presence of antibiotics selects for cells expressing amp R gene contains plasmid amp R gene codes for b-lactamase that degrades ampicillin, which normally kills bacteria

21 Bacterial transformation After treatment, only cells with the plasmid will grow on plates treated with ampicillin to eliminate recircularized vectors from further examination, lacz gene part of vector Insertion of chromosomal DNA disrupts lacz gene lacz codes for b-galactosidase which cleaves colorless X- Gal into a blue dye Recircularized plasmids will form blue colonies Recombinant vectors will form white colonies

22 BACTERIAL TRANSFORMATION

23 COLLECTING THE RECOMBINANT PRODUCT

24

25 Applied recombinant DNA technology RECOMBINANT PROTEIN PRODUCTS

26 RECOMBINANT DNA TECHNOLOGY HAS BEEN USED FOR MANY APPLICATIONS

27 Insulin made by recombinant bacteria Prior to 1982, insulin isolated from cattle Some people developed allergies and had to use cadaver insulin Insulin composed of 2 polypeptides A and B A and B coding sequence inserted into E.coli Fusion proteins extracted and β-galactosidase removed Purified A and B chain mixed to form functional protein

28 INSULIN PRODUCTION

29 Applied recombinant DNA technology CREATING GENETICALLY MODIFIED PLANTS

30 Why genetic modification? The world s population is expanding (some statistic on population expansion). To feed this growing population we need to find ways to increase food production/nutrition from the land we have Desirable traits for plants Drought and stress tolerance Disease resistance Improved nutritional value

31 AGROBACTERIAM IS A USEFUL TOOL FOR PLANT TRANSFORMATION Agrobacterium tumefaciens naturally infects plant cells and causes tumors Copyright Malcolm Storey Contains Ti plasmid that integrates into host chromosome Codes for plant growth hormones that form crown gall tumor

32 GENERATING CLONING VECTORS FOR PLANT TRANSFORMATION

33 GENE DELIVERY IN PLANTS

34 GENE DELIVERY IN PLANTS Vector mediated gene delivery via Agrobacterium Protoplast transformation Gene gun delivery

35 PLANT REGENRATION

36 Tissue regeneration Plants are recovered on media containing all of the nutrients required for plant growth (a carbon source, vitamins, macro and micro nutrients, selection) Plants are growth and then tested for the presence of the recombinant gene by PCR

37 Applied recombinant DNA technology GENETICALLY MODIFIED PLANTS

38 Direct transformation of rice genotypes with agronomically useful genes Gene Trait Reference psy, crt1, lyc Provitamin A biosynthesis Datta et al. (2003) ferritin Iron Storage Vasconcelos et al. (2003) FRO2 Resistance to insect pests Datta et al. (unpublished) Xa21 Bacterial leaf blight resistance Balchandrin et al. (2003), Narayanan et al. (2002, 2004) Chitinase Sheath blight tolerance Baisakh et al. (2001) Datta et al. (2001) enod12 Early nodulation Reddy et al. (2001) PEPC Increased photosynthesis Dtta et al. unpublished glgc Increased starch biosynthesis Datta et al. unpublished Adapted from table in Altpeter et al.

39 Bacterial blight caused by Xanthomonas oryzae pv. oryzae is one of the most destructive diseases of rice causes losses of up to 50% in areas of Asia Chemical control is not good at controlling the disease Xa21 is a bacterial blight resistance gene showing resistance to all known strains of Xanthamonas in India and the Phillipines Transformed indica rice line IR72 with Xa21

40 Xa21 is a bacterial blight resistance gene showing resistance to all known strains of Xanthamonas in India and the Phillipines. This gene was taken from a rice plant that is not commercially grown and cloned into a nonresistant rice plant that is used agriculturally. T = transgenic C = untransformed control Tu et al. (1998) Theor appl genet

41 Xa21 in field trials Genetically modified rice carrying Xa21 gene were tested in the field and compared with wildtype rice plants GM Rice Wildtype rice Results of field tests were impressive - A success story!

42 Case study: Glyphosate resistant plants Glyphosate = broad spectrum herbicide Inhibits enzymatic step in production of aromatic amino acid pathway in plants and bacteria A safe herbicide due to natural degradation in soil to harmless natural products Non-selective Transgenic crops resistant to Glyphosate would be very useful!

43 Concerns that herbicide resistance generated through nuclear transformation could escape through pollen dispersal into weeds Super resistant weeds

44 Daniell et al. (1998) Nature Biotechnology Cloned the EPSPS gene from petunia, conferring resistance to glyphosate into chloroplast expression vector Shoots of tobacco were transformed and regenerated Selected for spec/strep resistance Confirmed chloroplast gene insertion by PCR Seeds collected from 1 st self cross were grown and tested:

45 Gene therapy using a retroviral vector

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