Microbial Biotechnology

Chapter 5

The Structure of Microbes z Prokaryotes

• Archaebacteria • Includes halophiles, thermophiles, “extremophiles”

• Eubacteria • On skin, soil, water, can be pathogenic

The Structure of Microbes z Characteristics of Prokaryotes

• Generally smaller than Eukaryotes • No nucleus • Cell wall composed of peptidoglycan • Conjugation (transfer of DNA by cytoplasmic bridge) • Transduction (DNA is packaged in a virus and infects

recipient bacterial cells) • 20 minute growth rate (binary fission

Yeast are Important Too!

z Single celled eukaryote z Kingdom: Fungi z Over 1.5 million species z Source of antibiotics, blood cholesterol lowering

drugs z Able to do post translational modifications z Grow anaerobic or aerobic z 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

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

amylases

z Bacterial Transformation • The ability of bacteria to

take in DNA from their surrounding environment

• Bacteria must be made competent to take up DNA

Microorganisms as Tools

z Cloning and Expression Techniques • Fusion Proteins

Microorganisms as Tools

z Microbial Proteins as Reporters • Examples: the lux gene which

produces luciferase • Used to develop a fluorescent

bioassay to test for TB

Microorganisms as Tools

z Yeast Two-Hybrid System • Used to study protein interactions

Microorganisms as Tools

Using Microbes for a Variety of Everyday Applications

z 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)

Using Microbes for a Variety of Everyday Applications

z Food Products • Energy production in bacteria

• Aerobic or anaerobic

Using Microbes for a Variety of Everyday Applications

z Food Products • Fermentation (anaerobic respiration) • Lactic acid fermentation

• Used to make cheese, yogurt, etc.

• Ethanol fermentation • Used to make beer

and wine

z Therapeutic proteins • Recombinant insulin in bacteria

Using Microbes for a Variety of Everyday Applications

z 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 for a Variety of Everyday Applications

z Using Microbes Against Other Microbes • Antibiotics • Act in a few key ways

• Prevent replication • Kill directly • Damage cell wall or prevent its synthesis

Using Microbes for a Variety of Everyday Applications

Vaccines

z First was a vaccine against smallpox (cowpox provides immunity) • DPT-diphtheria, pertussis, and tetanus • MMR –measles, mumps, and rubella • OPV- oral polio vaccine (Sabin)

z 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)

Vaccines

Heavy chain

Light chain

IgA – first line of defense

IgG and IgM – activates macrophages

Vaccines

z Antigens stimulate antibody production in the immune system

Vaccines

z Mechanism of Antibody Action

z 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)

z What about flu vaccines (why do we have to get a shot every year?)

Vaccines

Bacterial and Viral Targets for Vaccines

z Immune Therapy • Used to enhance natural

immune responses • Ex: Interferon is a protein that

triggers production of protective proteins (or antibodies) that inhibit viral replication.

Bacterial and Viral Targets for Vaccines

z HIV

Microbial Genomes z 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

z Sequencing Strategies

Microbial Genomes

z Why study viral genomes? • Decipher genes and their products so that agents

that block attachment, block replication can be made

Microbial Diagnostics

z Using Molecular Techniques to Identify Bacteria • RFLP • PCR and Real time PCR • Sequencing

Microbial Diagnostics

z Microarrays for tracking contagious disease • PulseNet used to identify

outbreaks

z Combating Bioterrorism • 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

Microbial Diagnostics

z Using Biotech Against Bioweapons • Postal service x-raying packages • Antibody tests in the field • PCR tests in the field • Protein Microarrays for detecting bioweapon pathogens

Microbial Diagnostics