Microbial Biotechnology

Chapter 5

Fall 2006

The Structure of Microbes

Prokaryotes Archaebacteria

Includes halophiles, thermophiles, “extremophiles” Eubacteria

On skin, pathogens, soil, water Generally smaller than Eukaryotes (1-5um vs 10-100

um) What are some other characteristics of prokaryotes?

(cell wall (gram stain), no nucleus, binary fission, 20 min growth rate…)

Do you how to isolate single colonies? (Fig 5.2)

Yeast are Important Too! Single celled eukaryote Kingdom: Fungi Over 1.5 million species –only 10% have been

identified Source of antibiotics, blood cholesterol lowering

drugs Able to do post translational modifications Grow anaerobic or aerobic Examples: Pichia pastoris (grows to a higher

density than most laboratory strains), has a no. of strong promoters, can be used in batch processes

Microorganisms as Tools

Microbial Enzymes Taq (DNA polymerase), cellulases, proteases,

amylases Bacterial Transformation (figure 5.3)

The ability of bacteria to take in DNA from their surrounding environment

Cells must be made competent (to take up DNA)

Electroporation (figure 5.4) A mixture of bacteria and plasmid are briefly

electrically shocked

Cloning and Expression Techniques

Fusion Proteins (Figure 5.5) Use recombinant DNA methods to insert the

gene for a protein of interest into a plasmid containing a gene for a well-known protein that serves as a “tag”

The tag allows for isolation and purification Ex. His tagged GFP Affinity chromatography: Ni column binds to

repeated his amino acid tag

The Yeast Two-Hybrid System

Used to study protein interactions (figure 5.7) The gene for one protein of interest is cloned

and expressed as a fusion protein attached to the DNA-binding domain (DBD) of another gene (the bait). The gene for the second protein of interest is fused to another gene that contains transcriptional activator domain (AD) sequences (prey). If the two proteins interact then transcription occurs and the reporter gene product is expressed! (figure 5.7)

Microbial Proteins as Reporters

Examples: the lux gene which produces luciferase Used to develop a fluorescent bioassay to

test for TB (the lux gene is in a virus that only infects M. tuberculosis). If the bacteria is present, the virus infects the cells and the bacterial cells glow!

Using Microbes for a Variety of Everyday Applications

Food Products Rennin used to make curds (solid) and whey

in production of cheese Recombinant rennin is known as chymosin

(first recombinant food product approved by FDA)

Energy Production in Bacteria

Aerobic respiration (oxygen is final electron acceptor) Anaerobic respiration (inorganic molecules, such as

nitrate, sulfate, or carbonate, are final electron acceptors)

Fermentation/ anaerobic but doesn’t involve an electron transport chain (beers, wines yogurts etc.) Fig 5.8 (done by prokaryotes and eukaryotes)

Purpose: To produce NAD so that the organism can make ATP under anaerobic conditions (substrate level phosphorylation during glycolysis)

Glucose pyruvate (produce ATP and NADH) Two types: lactic acid and alcohol (NADH NAD) Pyruvate lactic acid or alcohol and carbon dioxide

Therapeutic proteins Recombinant insulin in bacteria (figure 5.9

and table 5.1) What is Type I diabetes (insulin-dependent

diabetes mellitus) Inadequate production of insulin by beta cells in

the pancreas

Field Applications of Recombinant Microorganisms

Ice-minus bacteria (remove ice protein producing genes from P. syringae)

P. fluorescens containing the gene that codes for the bacterial toxin from Bacillus thuringiensis (kills insects) Bt toxin!

Using Microbes Against Other Microbes Antibiotics (table 5.2 and figure 5.10) Penicillin was the first Act in a few key ways

Prevent replication Kill directly Damage cell wall or prevent its synthesis

How do antibiotic resistant strains arise? How can studying bacterial pathogens lead to

new drugs?

Vaccines

Figure 5.11 First was a vaccine against smallpox

(cowpox provides immunity) DPT-diphtheria, pertussis, and tetanus MMR –measles, mumps, and rubella OPV- oral polio vaccine (Sabin)

A Primer on Antibodies

Antigen- foreign substances that stimulate an immune response

Types of leukocytes or white blood cells B-lymphocytes: antibody-mediated

immunity T-lymphocytes: cellular immunity Macrophages: “cell eating” (phagocytosis)

                         

How are Vaccines Made?

They can be part of a pathogen (e.g. a toxin) or whole organism that is dead or alive but attenuated (doesn’t cause disease) Subunit (toxin) or another part of the pathogen Attenuated (doesn’t cause disease) Inactivated (killed)

What about HIV? What about flu vaccines (why do we have to get

a shot every year?)

Microbial Genomes

(Figure 5.15 and table 5.3) Microbial Genome Program (MGP) –the

goal is to sequence the entire genomes of microorganisms that have potential applications in environmental, biology, research, industry, and health

Sequencing Strategies

Viral Genomes

(table 5.4)- Examples of medically important viral genomes that have been sequenced Why?

Decipher genes and their products so that agents that block attachment, block replication can be made

Microbial Diagnostics

(figure 5.16) Using Molecular Techniques to Identify Bacteria RFLP PCR and Real time PCR

PulseNet (Contaminated food)

Combating Bioterrorism

(table 5.5) The use of biological materials as weapons to harm humans or animals and plants we depend on for food Examples in History

Throwing plague infected dead bodies over the walls of their enemies

Using Biotech Against Bioweapons Postal service x-raying packages Antibody tests in the field PCR tests in the field