Genetic Engineering

1. Genetic Engineering

2. What is Genetic Engineering? • Genetic Engineering = inserting a foreign gene of interest into a host to transcribe and translate a particular protein. • Ex. Inserting the human insulin gene into bacteria to mass produce it. Image taken without permission from http://www.medicalprogress.org/uploads/images/insulin%20inject%20WSU%20210.jpg

3. General Steps • Obtain the gene of interest (ex. insulin gene) • Insert the gene into the host (ex. bacteria) • Allow the host to multiply and express the foreign gene • get your desired protein! • Get lots of cells that can make the protein = clones

4. The Big Picture • The inserted gene is transcribed and translated using the RNA Polymerase, ribosomes and other resources in the cell

5. chromosomal DNA plasmids Plasmids • Circular DNA • Extrachromosomal • NOT part of the E. coli genome • “extra” DNA • Contain a few non-essential genes • Can give the bacteria additional “traits” • Depends on the genes on the plasmid • Can be exchanged between bacteria

6. Recombinant plasmids • Plasmids can be modified in biological labs • Modified plasmid = Recombinant plasmid • Plasmids can be used as cloning vectors to get the recombinant plasmid into E. coli • Cloning vectors = way to get the gene of interest into the host

7. Transformation • Process in which foreign DNA is physically inserted into host E. coli cells. • E. coli that contains recombinant plasmid = Transformed cell Image taken with out permission from http://summerschool.at/static/irismaria.schoenbrunner/images/transformation.png

8. Transformation Steps • Recombinant plasmids and host E. coli are mixed together • CaCl2 is added • The Ca2+ ions neutralize the negative charges on plasmid DNA • Help plasmid enter the membrane Image taken without permission from Transformation Animation. Available at http://www.dnai.org/b/index.html

9. Transformation Steps • Heat Shock • By rapidly changing the temperature of the solution, temporary pores are opened in the membrane • Creates an opening for the plasmids to enter the E. coli

10. Transformation • Transformation is not 100% successful • After transformation • Some cells will contain plasmid = transformed • Some cells won’t contain plasmid = untransformed • In a later step, you will determine which cells were transformed

11. E. Coli as a host • E. coli is a good host because: • Reproduce quickly (once every 20 minutes) • Nonpathogenic (the strain we use is not harmful) • Genome fully characterized (all genes have been sequenced)