Chapter 12 Lecture Outline Molecular Techniques and Biotechnology

Chapter 12Lecture Outline

Molecular Techniques and Biotechnology

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


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


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


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


Selection for Acid-Sensitive Mutants


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


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


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


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


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


Concept QuizHow 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.

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Chapter 12 Lecture Outline Molecular Techniques and Biotechnology