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Chapter 5 Microbial Biotechnology

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CHAPTER 5 MICROBIAL BIOTECHNOLOGY

BTEC3301

Microbial Biotechnology

MICROORGANISMS AS TOOLS INDUSTRIAL USE OF ENZYMES Several thermostable enzymes, like the Taq

polymerase have been identified and widely used in PCR and other reactions. Cellulase is obtained from E.coli and degrades

cellulose, a polysaccharide in plant cells.

Microbial Biotechnology

MICROORGANISMS AS TOOLSINDUSTRIAL USE OF ENZYMES

The denim jean is treated with cellulase, from

fungi Trichoderma reesei and Aspergillus niger, to give the faded look and texture. The protease subtilisin, from Bacillus subtilis,

forms component of Laundry detergent to remove and degrade protein stains.

Microbial Biotechnology

MICROORGANISMS AS TOOLSINDUSTRIAL USE OF ENZYMES

Enzymes can rightly be called the catalytic machinery of living systems. Enzymes are responsible for fermentation of sugar to ethanol by yeasts, a reaction that forms the bases of beer and wine manufacturing. Enzymes oxidize ethanol to acetic acid. This reaction has been used in vinegar production for

thousands of years.

Microbial Biotechnology

MICROORGANISMS AS TOOLS Similar microbial enzyme reactions of acid INDUSTRIAL USE OF ENZYMES forming bacteria and yeasts are responsible for aroma forming activities in bread making.

Presently more than 2000 different enzymes have been isolated and characterized. More than 75% of industrial enzymes are hydrolases. 40% of all enzyme sales are Protein-degrading enzymes .

Microbial Biotechnology

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Enzyme Production by Microbial Fermentation Extracellular enzymes are secreted outside the cell makes the recovery and purification process much simpler compared to production

of intracellular enzymes .

Microbial Biotechnology

MICROORGANISMS AS TOOLSEnzyme Production by Microbial Fermentation

Intracellular enzymes must be purified from

thousands of different cell proteins and other components. The organism producing the enzymes should

have a GRAS-status, which means that it is Generally Regarded as Safe. This is especially important when the enzyme produced by the organism is used in food processes .

Microbial Biotechnology

MICROORGANISMS AS TOOLSEnzyme Production by Microbial Fermentation

The organism should be able to produce high

amount of the desired enzyme in a reasonable time frame. The industrial strains typically produce over

50-g/l extracellular enzyme proteins. Most of the industrial enzymes are produced

by a relatively few microbial hosts like Aspergillus and Trichoderma fungi, Streptomyces and Bacillus .

Microbial Biotechnology

MICROORGANISMS AS TOOLSEnzyme Production by Microbial Fermentation

Most of the industrially used microorganisms

have been genetically modified to overproduce the desired activity and not to produce undesired side activities.

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Protein engineering Often enzymes do not have the desired

properties for an industrial application. E.g extreme thermo stability or overproduction of the enzyme.

Microbial Biotechnology MICROORGANISMS AS TOOLSProtein Engineering

Protein engineering is used to improve

commercially available enzyme to be a better industrial catalyst. Several enzymes have already been

engineered to function better in industrial processes. These include proteinases, lipases, cellulases and few amylases

Microbial Biotechnology MICROORGANISMS AS TOOLSProtein Engineering

Xylanase from fungus Trichoderma sps. is a good example of an industrial enzyme, used in pulp

and paper industry and needs to be stable in high temperature. Xylanases is a good example of engineered enzyme from Trichoderma. Its xylanase has been purified and crystallized. By designed mutagenesis its thermal stability has been increased about 2000 times at 70oC and its pHoptimum shifted towards alkaline region by one pH-unit.

Microbial Biotechnology

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Enzyme Technology Enzyme technology involves how to use

enzymes. The simplest way to use enzymes is to add them into a process stream where they catalyse the desired reaction and are gradually inactivated during the process .

Microbial Biotechnology MICROORGANISMS AS TOOLSEnzyme Technology

Example liquefaction of starch with amylases, bleaching

of cellulose pulp with xylanases or use of enzymes in animal feed. An alternative way to use enzymes is to immobilize them

so that they can be reused. One method of immobilization is to use ultrafiltration

membranes in the reactor system. The large enzyme molecules cannot pass the membrane but the small molecular reaction products can. Therefore enzymes are retained in a reaction system and the products leave the system continuously .

Reading Assignment for Quiz:Detergent, Food & BeveragesAnimal feed,personal care etc slide 15-40

Large scale enzyme applications Detergents Detergents were the first large scale application for microbial enzymes. Bacterial proteinases are still the most important detergent enzymes. Some products have been genetically engineered to be more stable in the

hostile environment of washing machines with several different chemicals present.

Microbial Biotechnology MICROORGANISMS AS TOOLSLarge scale enzyme applicationsDetergents

Lipid degrading enzymes, lipase, were used in

powder and liquid detergents to decompose fats. Lipase is produced in large scale by Aspergillus

oryzae host after cloning the Humicola gene into this organism. Amylases are used in detergents to remove

starch based stains.

Microbial Biotechnology MICROORGANISMS AS TOOLSLarge scale enzyme applicationsDetergents

Cellulases have been part of detergents since

early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose. In textile cellulases remove cellulose microfibrils, which are formed during washing. Alkaline cellulases are produced by Bacillus

strains and neutral and acidic cellulases by Trichoderma and Humicola fungi.

Microbial Biotechnology

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Large scale enzyme applicationsFoods/Beverages produced by Microbial Activity

Yogurt, cheese, chocolate, butter, pickles, sauerkraut, soy sauce, food supplements (such as vitamins and amino acids), food thickeners

(produced from microbial polysaccharides), alcohol (beer, whiskeys, wines) and silage for animals are all products of microbial activity.

Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity

The industrial microbiologist/ biotechnologist

may be involved in producing concentrated microbial inocula for fermentations or the maintenance of fermentation systems utilized in production facilities. The use of starch degrading enzymes,

amylase, was the first large-scale application of microbial enzymes in food industry.

Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity

Enzymes have many applications in drink

industry. The use of chymosin in cheese making to coagulate milk protein. Another enzyme used in milk industry is beta-

galactosidase or lactase, which splits milksugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers.

Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity

Enzymes are used also in fruit juice

manufacturing. Pectins are substances in fruit lamella and cell walls. The cell wall contains also hemicelluloses and cellulose. Pectinase, xylanase and cellulase improve the liberation of the juice from the pulp. Pectinases and amylases are used in juice clarification.

Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity

Brewing is an enzymatic process. Malting is a

process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide. Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the

yield.

Microbial Biotechnology MICROORGANISMS AS TOOLS Large scale enzyme applications Foods/Beverages produced by Microbial Activity

Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes, amylase, are liberated and they hydrolyse (Break

down) the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide. Similarly enzymes are widely used in wine production to obtain a better extraction of the necessary components and thus improving the

yield.

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Large scale enzyme applicationsFoods/Beverages cured or improved by microbial activity Production of coffee, tea, cocoa, summer sausage,

vanilla, cheese, olives and tobacco all require

microbial activity.

Microbial Biotechnology

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Large scale enzyme applicationsFood flavoring agents and preservatives

Organic acids such as citric, malic and ascorbic

acids and monosodium glutamate are microbialproducts commonly used in foods.

Microbial Biotechnology

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Large scale enzyme applications

Foods Mushrooms, truffles and some red and green algae

are consumed directly. Yeasts are used as food

supplements for humans and animals.

Microbial Biotechnology

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Large scale enzyme applications

Oil recovery/mining Oil recovery may be facilitated by the development of unique bacteria which produce a surfactant that forces trapped oil out of rocks. Extraction of minerals from low-grade ores is

enhanced by some bacteria (microbial leaching).

Microbial Biotechnology

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Large scale enzyme applications Waste and Wastewater Management Isolating or developing microbial strains capable of degrading and detoxifying hydrocarbon and halogenated hydrocarbon waste (for example

organophosphates, acetylcholinesterase inhibitors) of industrial, agricultural or military origin is essential in waste management.

Microbial Biotechnology

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Large scale enzyme applications

Textiles The use of enzymes in textile industry is one of the

most rapidly growing fields in industrial enzymology. Starch has for a long time been used as a

protective glue of fibers in weaving of fabrics. This is called sizing.

Enzymes are used to remove the starch in a process called desizing. Amylases are used in this process since they do not harm the textile fibers. Laccase a polyphenol oxidase from fungi is used

MICROORGANISMS AS TOOLS Large scale enzyme applications Textiles

to degrade lignin the aromatic polymer found in all plant materials .

Microbial Biotechnology

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Large scale enzyme applications

Animal Feed The net effect of enzyme usage in feed has been increased animal weight. The first commercial success was addition of beta-

glucanase into barley based feed diets. Barley contains beta-glucan, which causes high viscosity in the chicken gut.

Xylanase, from Trichoderma, are added to wheatbased broiler feed and are nowadays routinely used in feed formulations and animals gain

MICROORGANISMS AS TOOLS Large scale enzyme applications Animal Feed

weight. Enzymes have become an important aspect of

animal feed industry. In addition to poultry, enzymes are used in pig feeds and turkey feeds.

Microbial Biotechnology

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Large scale enzyme applications

Baking Alpha-amylases have been most widely studied in

connection with improved bread quality and increased shelf life. Both fungal and bacterial amylases are used in

bread making and excess may lead to sticky dough.

Microbial Biotechnology

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Large scale enzyme applications

Pulp and Paper The major application is the use of

xylanases in pulp bleaching for paper.

Microbial Biotechnology

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Large scale enzyme applications

Leather Leather industry uses proteolytic and

lipolytic enzymes in leather processing. Enzymes are used to remove animal skin,

hair, and any unwanted parts.

Microbial Biotechnology

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Large scale enzyme applications

Leather The used enzymes are typically alkaline

bacterial proteases. Lipases are used in this phase or in bating

phase to specifically remove grease.

Microbial Biotechnology

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Large scale enzyme applications Enzymes in Personal Care products

Personal care products are a relatively new area for enzymes and the amounts used are small but worth to mention as a future growth area.

Microbial Biotechnology

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Large scale enzyme applications

Enzymes in Personal Care products One application is contact lens cleaning. Proteinase and lipase containing enzyme solutions are used for this purpose. Hydrogen peroxide is used in disinfections of contact lenses. The residual hydrogen peroxide after disinfections can be removed by a heme containing catalase enzyme, which degrades hydrogen peroxide.

Microbial Biotechnology

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Large scale enzyme applicationsEnzymes in Personal Care products Some toothpaste contains glucoamylase and

glucose oxidase. Dentures can be cleaned with protein degrading enzyme solutions. Enzymes are also used for applications in skin and

hair care products.

Microbial Biotechnology

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Large scale enzyme applications

Enzymes in DNA-technology DNA-technology has revolutionized both traditional biotechnology and opened totally new fields for scientific study. Recombinant DNA-technology allows one to

produce new enzymes in traditional overproducing and safe organisms .

Microbial Biotechnology

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Large scale enzyme applicationsEnzymes in DNA-technology

Protein engineering is used to modify and improve existing enzymes. DNA is basically a long chain of deoxyribose sugars

linked together by phosphodiester bonds. Organic bases, adenine, thymine, guanine and cytosine are linked to the sugars and form the alphabet of genes. The specific order of the organic bases in the chain constitutes the genetic language.

Microbial Biotechnology

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Large scale enzyme applicationsEnzymes in DNA-technology Genetic engineering means reading and modifying

this language. Enzymes are crucial tools in this process. E.g.:

Microbial Biotechnology

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Large scale enzyme applicationsEnzymes in DNA-technology

Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process.

E.g.:

1. Restriction enzymes recognise specific DNA sequences and cut the chain at these recognition sites.2. DNA modifying enzymes synthesize nucleic acids,

degrade them, join pieces together and remove parts of the DNA.

3. DNA-polymerases synthesize new DNA-chains. Many of them need a model template, which they copy. 4. Ligases join adjacent nucleotides together.

Microbial Biotechnology

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Therapeutic Proteins by Gene Transfer Recombinant DNA technology led to the rapid

development and production of Therapeutic protein. There are many proteins essential to good

health that some people cannot produce because of genetic defects.

Microbial Biotechnology

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Therapeutic Proteins by Gene Transfer These proteins include various blood-clotting

factors causing hemophilia, insulin (resulting in diabetes), growth hormone (resulting in lack of proper growth), and other proteins, the administration of which corrects pathological conditions or results in other therapeutic benefits. Plasmids are used to transfer human genes to bacterial cells.

Microbial Biotechnology

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Therapeutic Proteins by Gene Transfer If the gene inserted into the plasmid of bacteria is

the human gene for insulin, for example, the bacteria into which this gene is inserted produces human insulin. Bacteria as such do not produce insulin, but the recombinant bacterial cells do produce insulin, it was an outstanding example of microbial biotechnology.

Courtesy John J. Cardamone, Jr.

Insertion of a DNA section into a plasmid

Microbial Biotechnology

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Therapeutic Proteins by Gene Transfer

cDNA: Human genes composed of coding and noncoding sequences. The copy of the coding sequences is called cDNA. The synthesis of the insulin cDNA will allow the production of a functional insulin molecule.

Transfer of the Insulin gene into a plasmid vector (schematic)

Microbial Biotechnology

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Therapeutic Proteins by Gene Transfer

Cloning the Insulin gene (Mechanism): Insulin was first synthesized in 1979 in E. coli

cells through the use of recombinant DNA techniques. Insulin is produced by beta cells in pancreas in

humans.

Microbial Biotechnology two polypeptides subunits Human insulin has MICROORGANISMS AS TOOLSTherapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):

called the A (21 amino acids) and the B (30 amino acids) which are bonded by disulphide bond to create the active insulin.

When a human gene for insulin is cloned, gene

for each of the subunit is inserted into plasmid vector separately (Fig 5.9).

MicrobialvectorTOOLS the Lac z gene encoding for the The Biotechnology MICROORGANISMS AS hasTherapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):

enzyme -galactosidase (-gal).

The genes that code for the two insulin chains in

human are fused to the E. coli gene (Lac z) encoding for beta-galactosidase. The plasmid is then transformed into E.coli . Plasmids enter the bacteria in a process called transfection .

Microbial Biotechnology With the AS TOOLS MICROORGANISMS recombinant DNA molecule successfullyTherapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):

inserted into the bacterial host, another property of plasmids can be exploited - their capacity to replicate.

Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies. Because the insulin genes are connected to the lac z gene, when bacteria synthesizes proteins from these plasmids, they produce a protein containing -gal attached to human insulin protein and this is called a fusion protein .

MicrobialfusionTOOLS The Biotechnology MICROORGANISMS AS protein here is called -gal-insulinTherapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):

fusion protein.

When the bacteria divide, the plasmids are

divided between the two daughter cells and the plasmids continue to reproduce. With cells dividing rapidly (every 20 minutes), a

bacterium containing human cDNA) will shortly produce many millions of similar cells (clones) containing the same human gene.

Microbial Biotechnology After the chains are synthesized, the bonds that MICROORGANISMS AS TOOLSTherapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):

hold the insulin molecule to the betagalactosidase are cleaved with cyanogen bromide.

Affinity column is used to separate the two proteins. The two chains are then purified to give native

insulin. This form of insulin is an exact match to that which is made in the body.

Fig 5.9: Using bacteria to produce Human insulin

NEW & VIEWSType 1 or insulin-dependent diabetes mellitus & study steps in Fig.5.9.

Microbial Biotechnology

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Therapeutic Proteins by Gene Transfer

Microbes against microbes Antibiotics are antimicrobial drugs used

against microbes. An antibiotic is a substance, usually produced

by a microorganism which, in very small quantities, inhibits or kills other microorganisms .

Microbial Biotechnology

MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer Microbes against microbes

Both natural and chemically enhanced microbial

products can be used to control human, animal and plant diseases. Using traditional genetics or recombinant DNA

techniques, the microorganism can be modified to improve the yield or action of antibiotics and other antimicrobial agents.

Microbial Biotechnology

MICROORGANISMS AS TOOLS Therapeutic Proteins by Gene Transfer Microbes against microbes

New research directions are aimed at discovering microbial metabolites with pharmacological activities useful in the

treatment of hypertension, obesity, coronary heart disease, cancer and inflammation.

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VaccinesIntroduction A number of diseases are caused by microorganisms Vaccines are essential to protect humans and animals from microbial diseases. Recombinant DNA technology has allowed the production of

novel vaccines that offer protection without the risk of infection (e.g. hepatitis B vaccine).

Microbial Biotechnology

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Vaccines: Introduction

For many bacterial diseases, and some fungal

diseases, there are antibiotics, produced by other micro-organisms. There are very few means of fighting viral

diseases. The production of vaccines against the microbial pathogens, and more particularly the pathogenic viruses, in order to immunise the susceptible populations, is a safe and more certain recourse.

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Vaccines: Introduction

Biotechnology has made it now possible to

produce immunological agents to afford protection from diseases to large numbers of people. This area is immunotechnology, an arm of

biotechnology.

Microbial Biotechnology

MICROORGANISMS AS TOOLSVaccines

What are Vaccines A vaccine is an agent, sourced from the pathogen, and is deliberately introduced into the

mammalian system in order to impart a memory of the pathogen or its pathogenic component The memory is imparted on the first contact of the vaccine with the mammalian immune system.

Microbial Biotechnology

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Vaccines: What are Vaccines

Vaccines contain antigens (that elicit the

production of antibodies), or immunogens (that trigger the cellular component of immune response) In the event of an encounter with the

corresponding antibodies, only the antigens can bind with the antibodies, and form an antigenantibody complex that neutralises the harmful effects of the antigens or the organisms that produce them.

MICROORGANISMS AS TOOLSVaccines

Microbial Biotechnology

Vaccination/Immunisation The process of the deliberate introduction of a vaccine into the organism is vaccination, for which the term inoculation is also often

used. Since vaccination immunises the organism, the process is also called immunisation When an organism is vaccinated, the immune system is readied to show an immune response by way producing antibodies against the pathogen.

MICROORGANISMS AS TOOLS Vaccines

Microbial Biotechnology

Composition of vaccines Vaccines are suspensions, in saline , of

weakened pathogenic organisms or the proteins they secrete, which have the potential to cause a disease.

MICROORGANISMS AS TOOLS Vaccines

Microbial Biotechnology

Types of vaccines:Inactivated vaccines: The pathogen is killed using heat or formalin, as for example, typhoid vaccines .

Attenuated vaccines: Vaccines: Types of vaccines: The pathogen is weakened (attenuated) by aging or altering growth conditions, but is alive, as in the case of measles, mumps and rubella vaccines .MICROORGANISMS AS TOOLS

Microbial Biotechnology

Avirulent organisms: A non-pathogenic strain of a pathogenic organism is used as a vaccine, as in BCG (Bacillus Calmette Guerin) vaccine against Mycobacterium tuberculosis, the tuberculosis bacterium.

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Toxoids:

Vaccines: Types of vaccines:

The toxin from the pathogen is used as an

antigen to produce the vaccine .

Acellular vaccines: Only the antigenic component of the organism

is used instead of the whole organism, as in Haemophilus influenza B vaccine .

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Vaccines: Types of vaccines:

Subunit vaccines: Genetic engineering techniques have now made

it possible to use as a vaccine only a part of an organism that is adequate to stimulate the immune response e.g in Hepatitis B vaccine a segment of genetic material is isolated from the pathogens and introduced into bacteria or yeasts .

DNA vaccines: Vaccines: Types of vaccines:MICROORGANISMS AS TOOLS

Microbial Biotechnology

DNA vaccines are an offshoot of gene therapy Selected segments of DNA, when introduced into the patients system synthesise and deliver proteins that are needed to replace the defective enzyme system or tag a cell for destruction. Viruses or lipid vehicles are used to deliver the DNA into the cells. This recent technology is being tried to produce vaccines against HIV, by a direct injection of plasmid borne DNA

2-WAY- LEARNING Hepatitis B vaccine? Immunisation by DNA injection?

Bioterrorism (Chapter 9)

REFERENCES:

Introduction to Biotechnology by W.J. Thieman and M.A. Palladino. Pearson & Benjamin Cummings 2nd edition. http://en.wikipedia.org Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen

Laboratory of Bioprocess Engineering, Helsinki University of Technology, FinlandHans Schoemaker, DSM Research, MD Geleen, The Netherlands http://www.accessexcellence.org/RC/VL/GG/transfer_and.html

PRODUCTION OF THERAPEUTIC PROTEINS BY GENETIC ENGINEERING - IMPACT No. 299 May 1998 Duane T. Ghttp://www.fbae.org/Channels/biotech_in_medicine/vaccines.htmish