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Feb 21, 2013 (4 years and 5 months ago)

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Chapter 12

Lecture 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


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 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

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

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.