1. Genetic Engineering An Overview

2. What is it??? Applied techniques of genetics and biotechnology (“Wet lab procedure”). Much trial and error. Applied techniques of genetics and biotechnology (“Wet lab procedure”). Much trial and error. Involves the “isolation, manipulation and reintroduction of DNA into cells or model organisms, usually to express a protein’’. Involves the “isolation, manipulation and reintroduction of DNA into cells or model organisms, usually to express a protein’’. DNA taken from one organism and inserted (transformed) into another (transgenic) organism DNA taken from one organism and inserted (transformed) into another (transgenic) organism Heritable, directed alteration of an organism. Altering DNA or adding new DNA allows us to change the characteristics of a cell or cells. Heritable, directed alteration of an organism. Altering DNA or adding new DNA allows us to change the characteristics of a cell or cells.

3. Why do it? It aims to introduce new characteristics or attributes physiologically or physically, such as making a crop resistant to a herbicide, introducing a novel trait, or producing a new protein or enzyme. It aims to introduce new characteristics or attributes physiologically or physically, such as making a crop resistant to a herbicide, introducing a novel trait, or producing a new protein or enzyme. For example… For example…

4. How does it work? Detect gene within organism of interest Detect gene within organism of interest Isolate and “Splice out” DNA Isolate and “Splice out” DNA “Ligate” into “Vector” of choice “Ligate” into “Vector” of choice Introduce into bacteria Introduce into bacteria Isolate colonies containing vector with gene of interest Isolate colonies containing vector with gene of interest Grow up bacteria colony to produce multiple copies of gene which is expressed Grow up bacteria colony to produce multiple copies of gene which is expressed

5. Isolating and splicing DNA of Interest… Conventionally entire genome fragmented using “Restiction Enzymes” Conventionally entire genome fragmented using “Restiction Enzymes” –E.g. BamH1 Hindiii Hindiii –Target Palindromic sequences: AGGTACCT AGGTACCT TCCATGGA TCCATGGA

6. –Can result in “Staggered”/”Sticky” ends AG GTACCT TCCATG GA –Can result in “Blunt” ends AGGT ACCT AGGT ACCT TCCA TGGA TCCA TGGA –Enzymes can create sticky ends from blunt ones

7. PCR can be used to specifically target gene of interest PCR can be used to specifically target gene of interest –“Enzymatic amplification of specific DNA fragment using repeated cycles of DNA denaturation, primer annealing and Chain extension” –Knowledge of full sequence not required –Can produce large amount of copies from >minute quantity of target DNA >Partially damaged DNA >Partially damaged DNA

9. Inserting Gene fragments into Vector of choice Many types of Vectors depending on needs Many types of Vectors depending on needs –Bacterial Artificial Chromosomes –Yeast Artificial Chromosomes –Plasmids Use RE to create complimentary sticky ends in the vector Use RE to create complimentary sticky ends in the vector

10. Possible outcomes post digestion with R.E. Possible outcomes post digestion with R.E. –Plasmid reforms –Fragmented genomic DNA joins up with itself –Plasmid and fragmented DNA for a hybrid Contains gene of interest Contains gene of interest Contains other gene (unfortunately, most times!!!) Contains other gene (unfortunately, most times!!!)

11. Insert into Bacteria/Host Allows expression of protein product Allows expression of protein product Allows multiple copies of the gene to be made Allows multiple copies of the gene to be made Several methods Several methods –Heat shock –Electric Pulse (electroporation) –Microinjection/microprojectiles –Viruses –Lysosomes

12. Identifying the right colony 3 levels of identification: 3 levels of identification: –Bacteria which have taken up a plasmid –Bacteria which have taken up a recombinant plasmid –Bacteria containing the wanted gene Achieved by using antibiotic resistence genes and gene probes. Achieved by using antibiotic resistence genes and gene probes.

13. Finally… Once the correct colony is identified it is cultured and gene expression encouraged. Once the correct colony is identified it is cultured and gene expression encouraged. Protein product can be harvested! Protein product can be harvested! However… it is not always successful… Much patience is required!!! However… it is not always successful… Much patience is required!!!

14. Some examples of current applications of genetic engineering Synthesis of insulin. Not always effective due to a eukaryotic protein being expressed in a prokaryotic cell. Synthesis of insulin. Not always effective due to a eukaryotic protein being expressed in a prokaryotic cell. Introduction of resistance genes in crop plants Introduction of resistance genes in crop plants Flavr Savr Tomato Flavr Savr Tomato Gene therapy (e.g. Cystic Fibrosis) Gene therapy (e.g. Cystic Fibrosis)