2. Genetic Engineering

3. 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

4. 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

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

6. 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 chromosomal DNA plasmids

7. 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

8. 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/imag es/transformation.png

9. Transformation Steps Recombinant plasmids and host E. coli are mixed together CaCl 2 is added –The Ca 2+ 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

10. 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

11. 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

12. 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)