Upload
katrina-villa
View
27
Download
0
Tags:
Embed Size (px)
DESCRIPTION
Biology 331- Chapter 12 Ppt
Citation preview
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
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
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 geneSequence 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
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
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. 6
Selection for Acid-Sensitive Mutants
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
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
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
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
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