1. Chapter 12 Lecture Outline Molecular Techniques and Biotechnology

2. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 2 Biotechnology Use of living organisms or their products to improve human health Began 10,000 years ago…  …yeast usage for bread, cheese, yoghurt, and alcohol (benefit?) production 1940s  Fleming discovered penicillin Today  recombinant production of human proteins, biopolymers, genetic engineering to transfer disease resistance and much more

3. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 3 Basic Tools of Biotech Bacteria grow quickly E. coli genome relatively well understood  Can activate transcription upon stimulation Small plasmids replicate quickly Insert gene of interest into a plasmid  Use restriction enzymes and ligases  Make huge amounts of protein  Easy to purify for further use Medicine  Recombinant human growth hormone and insulin Reagents for diagnostics and research Vaccine components Applied Biotechnology

4. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 4 Genetic Analyses Create mutants  Identify mutant phenotype E.g., loss of ability to grow at low pH Tagged with antibiotic resistance marker Identify mutated gene  Sequence to determine mutation Or locate site of larger deletion, insertion  Transposon—large insertion, easy to identify  Sequence DNA interrupted by mutation Compare to E. coli genome to identify gene

5. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 5 Genetic Analysis with Transposons Transposons easy to identify  Contain antibiotic-resistance genes Cells with transposons grow with antibiotics present  Create large insertion mutations Knock out gene function (loss of function)  Easy to identify sequence Use probe complementary to transposon sequence Mutated gene is located next to transposon

6. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 6 Selection for Acid-Sensitive Mutants

7. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 7 DNA Sequencing of Insertion Sites Design oligonucleotide primers that anneal to end of the transposon Sequence across the transposon into the adjacent DNA Perform computer based homology search with the known sequenced genome Most successful with completely sequences organisms

8. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 8 Exploring Gene Regulation: Reporter Fusions Promoters determine where, when genes are expressed Link promoter to reporter gene on plasmid  Easily observed protein product lacZ turns blue, gfp fluoresces green  Transform plasmid into cell  Determine what conditions allow transcription (turns cell blue or fluorescent)  Reveals transcriptional control only Link promoter + coding region to reporter  Translational fusion  Tells where protein is in cell  Reveals transcriptional and translational control

9. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 9 Applied Microbial Biotechnology Use microbes to express foreign proteins in large amounts  Eukaryotic proteins Human insulin Research tools  Diagnostic tools HIV antigens for ELISA  Vaccine components Pertussis toxin, HPV capsid Use microbial gene products  Microbes have widest diversity of enzymes PCR depends on archaeal polymerases Engineer genes into plants to improve shelf life and resistance against infections Viral gene therapy

10. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 10 B. thuringensis Crop Protection Bacterial protein is toxic to insects  Crystals protect bacterial spores  Harmless to animals Clone Bt gene into crops  Expressed in leaves of plant  Kills only insects that feed on those crops  Eliminates need to spray chemical pesticides Chemicals are dangerous to use Toxic to humans Expensive

11. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 11 Viral Gene Therapy Clone desired gene into viral genome  Replace gene necessary for pathogenic growth Infect person with modified virus  Infected cells will express cloned gene Make desired protein  Not all cells are infected Useful for proteins needed in only a few cells  Genetic diseases, individual not expressing needed protein  Integrate gene into chromosome Enhance recombination to replace defective gene Cure disease permanently

12. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 12 Concept Quiz How can you easily identify bacterial cells that have a transposon insertion? a. The cells’ DNA includes transposon sequence. b. The cells have an insertion in a gene. c. The cells are resistant to an antibiotic.