Biotechnology
Contents
The nature of biotechnology
Recombinant DNA Technology
Bioprocess/fermentation technology
Enzyme technology
Biological fuel generation
Definition
Definition:
the term "biotechnology" refers to the use of living organisms or their products to modify human health and the human environment.
Other definitions:
1. Biotechnologists use engineering and science to create new products from biologically based raw materials, such as vaccines or foods. They also develop factory processes to reduce pollution or treat waste products.
2. Biotechnology uses living cells and materials produced by cells to create pharmaceutical, diagnostic, agricultural,
environmental, and other products to benefit society
Periods of Biotechnology History
Pre- 1800: Early applications and speculation
1800-1900: Significant advances in basic
understanding
1900-1953: Genetics
1953- 1976: DNA research, science explodes
1977- present: modern biotechnology
Biotechnology
Genetic Engineering
– DNA cloning
• Isolation of DNA from one organism and
putting it into another
• Use of restriction enzymes
– Plasmid
– Integration in host DNA
• Use of bacteria (E. coli)
• Use of viral vectors
Uses of Technology
Protein production
Pharmaceutical proteins (insulin)
Vaccines
– Surface proteins
– DNA fragments that code for proteins
Pest resistant plants
Herbicide resistant plants
– Plants with improved nutritional value
– Edible vaccines
branches of biotechnology
Red biotechnology is applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures through genomic manipulation.
Green biotechnology is biotechnology applied to agricultural processes. An example would be the selection and domestication of plants via micropropagation. Another example is the designing of transgenic plants to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides.
White biotechnology , also known as industrial biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals.
Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare.
Bioremediation and Biodegradation
Biotechnology is being used to engineer and adapt organisms especially microorganisms in an effort to find sustainable ways to clean up contaminated environments.
Biological processes play a major role in the removal of contaminants and biotechnology is taking advantage of the astonishing catabolic versatility of microorganisms to degrade/convert such compounds.
Cloning
Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed.
There are two types of cloning:
– Reproductive cloning
– Therapeutic cloning
– Ian Wilmut, February 1997,
cloning of a sheep, named Dolly, from the mammary glands of an adult female
ENZYMES USED IN CLONING
– Restriction Enzymes
– Ligase
– DNA Polymerases
* E. coli DNA Polymerase I (The Klenow Fragment)
* Bacteriophage T4 and T7 polymerase
* Reverse Transcriptase
* RNA Polymerases
– Phosphatase and Kinase
Techniques and Strategies of Gene Cloning
Type I restriction enzymes
cut the DNA a thousand or more base pairs away from the recognition site. This is done by looping the DNA around so that the enzyme binds both at the recognition site and the cutting site.
Each molecule of a type I restriction enzyme can cut DNA only a single time and then it is inactivated!
Type II restriction enzymes
Type II restriction enzymes cut the DNA within the recognition sequence. Some generate blunt ends, others give sticky ends.
Sticky ends are more convenient than blunt ends when joining together fragments of DNA using DNA ligase.
When two sticky ends made by the same enzyme are ligated, the junction may be cut apart later by using the same enzyme again. However, if two sticky ends made by two different enzymes are ligated together, a hybrid site is formed that cannot be cut by either enzyme
Type II Restriction Enzymes: Blunt Versus Sticky Ends
Matching of Compatible Sticky Ends
Restriction Enzymes
Making a Restriction Map
A restriction map is a diagram showing the location of cut sites on DNA for a variety of
restriction enzymes.
Restriction maps are deduced by cutting the target DNA with a selection of restriction enzymes, both alone and in pairs.
Restriction Mapping
DNA Fragments are Joined by DNA Ligase
DNA Polymerases
DNA Probe
Bacteriophage T4 and T7 polymerase
Reverse Transcriptase
Phosphatase and Kinase
TECHNIQUES USED IN CLONING
DNA Isolation
Gel Electrophoresis
– Agarose Gel Electrophoresis
– Polyacrylamide Gel Electrophoresis
Southern Transfer
Western Blot
Colony Blot
TECHNIQUES USED IN CLONING
Hybridization
Immunological Techniques
Polymerase Chain Reaction
DNA Sequencing
Site-Directed Mutagenesis
Non-radioactive Detection Methods
Isolation of DNA
Modern technology has led to a steady decrease in the amount of DNA needed for analysis.
Cell walls and membranes must be broken down to liberate the DNA from the cell.
Purification of DNA
Macromolecules such as DNA may be separated from smaller molecules by centrifugation.
Phenol dissolves proteins and is used to remove them from DNA.
Nucleic acids may be purified on columns containing resins that bind DNA and RNA.
Phenol Extraction Removes Proteins from Nucleic Acids
Removal of RNA by Ribonuclease
Agarose:
A polysaccharide from seaweed that is used to form gels for separating nucleic acids by electrophoresis.
Polyacrylamide:
Polymer used in separation of proteins or very small nucleic acid molecules by gel electrophoresis
Electrophoresis:
Movement of charged molecules due to an electric field. Used to separate and purify nucleic acids and proteins
gel electrophoresis:
Electrophoresis of charged molecules through a gel meshwork in order to sort them by size
agarose gel electrophoresis:
Technique for separation of nucleic acid molecules by passing an electric current through a gel made of agarose.
Gel Electrophoresis of DNA
Agarose Gel Electrophoresis of DNA
Agarose Gel Electrophoresis
DNA can be detected by radioactive labeling or by staining with a dye such as ethidium bromide.
Pulsed Field Gel Electrophoresis (PFGE)
Type of gel electrophoresis used for analysis of very large DNA molecules and which uses an electric field of “pulses” delivered from a hexagonal array of electrodes.
Pulsed Field Gel Electrophoresis
Denaturing gradient gel electrophoresis (DGGE)
Combination of gel electrophoresis with DNA denaturation that allows separation of DNA molecules differing in sequence by only a single base.
Small pieces of DNA that differ by as little as one base pair may be separated by this technique.
Denaturing Gradient Gel Electrophoresis
Resolved protein bands in polyacrylamide gel after electrophoresis and coomassie blue staining
Hybridization of DNA and RNA
Annealing: The rejoining of separated single strands of DNA to form a double helix.
hybrid DNA: Artificial double-stranded DNA molecule made by two single strands from two different sources.
Hybridization: Formation of double-stranded DNA molecule by annealing of two single strands from two different sources.
probe molecule: Molecule that is tagged in some way (usually radioactive or fluorescent) and is used to bind to and detect another molecule.
Melting of DNA at High Temperature
Southern, Northern, and Western Blotting
Northern blotting: Hybridization technique in which a DNA probe binds to an RNA target molecule
Southern blotting: A method to detect single stranded DNA that has been transferred to nylon paper by using a probe that binds DNA
Western blotting: Detection technique in which a probe, usually an antibody, binds to a protein target molecule
Southern Blotting: DNA:DNA Hybridization
Using probes to detect DNA sequences by hybridization can be carried out on a membrane and is then referred to as “blotting”.
Southern blotting requires the target DNA to be cut into smaller fragments and run on an agarose gel. The fragments are denatured chemically to give single strands, then transferred to a nylon membrane. Notice that the DNA is invisible both in the gel and on the membrane.
A radioactive probe (also single-stranded) is passed over the membrane. When the probe DNA finds a related sequence, a hybrid molecule is formed.
Southern Transfer
Detection of Radio-Labeled DNA
scintillation counting: Detection and counting of individual microscopic pulses of light.
Autoradiography: Allowing radioactive materials to take pictures of themselves by laying them flat on photographic film.
Scintillation counters measure radioactivity in liquid samples whereas autoradiography is used to locate radioactive molecules on gels or membranes.
Scintillation Counter is Used to Measure Radioactivity
Autoradiography to Detect Radio-Labeled DNA or RNA
Zoo Blotting Reveals Coding DNA
Zoo blotting: Comparative Southern blotting using DNA target molecules from several different animals to test whether the probe DNA is from a coding region.
Restriction Fragment Length Polymorphisms (RFLPs)
restriction fragment length polymorphism (RFLP): A difference in restriction sites between two related DNA molecules that results in production of restriction fragments of different lengths. When these are separated on a gel, bands of different sizes will appear.
Polymerase Chain Reaction (PCR)
PCR consists of cycles of repeating 3 steps: denaturation, annealing and polymerization.
PCR Machine or Thermocycler
Fundamentals of the Polymerase Chain Reaction
The PCR allows trace amounts of a DNA sequence to be amplified giving enough DNA for cloning, sequencing or other analyses.
PCR needs primers to start DNA synthesis which means that we must know some DNA sequence in or close to the region of interest.
PCR is a procedure involving multiple cycles of DNA strand separation, binding of primers, and synthesis of new DNA.
Denaturing the Template and Binding the Primers
Taq polymerase has high polymerase activity, contains no 3'→5‘ exonuclease activity, and is resistant to denaturation at higher temperatures than the E. coli enzyme
Elongation of New Strand by Taq Polymerase
The Second Cycle of the PCR
Degenerate Primers
Degenerate primer: Primer with several alternative bases at certain positions.
Degenerate primers are used when partial sequence information is available, but the complete sequence is unknown.
Degenerate DNA primers must also be used if only a protein sequence is available.
Inverse PCR
Method for using PCR to amplify unknown sequences by circularizing the template molecule.
TA Cloning by PCR
TA cloning: Procedure that uses Taq polymerase to generate single 3'-A overhangs on the ends of DNA segments that are used to clone DNA into a vector with matching 3'-T overhangs.
Randomly Amplified Polymorphic DNA (RAPD)
Randomly Amplified Polymorphic DNA, or RAPD, is usually found in the plural as RAPDs and is pronounced “rapids,” partly because it is a quick way to get a lot of information about the genes of an organism under investigation.
The purpose of RAPDs is to test how closely related two organisms are.
The principle of RAPDs is statistically based. Given any particular five-base sequence, such as ACCGA, how often will this exact sequence appear in any random length of DNA?
PCR may be performed with arbitrary primers. Comparing results from two samples of DNA reveals their relatedness.
Randomly Amplified Polymorphic DNA
Identification of Fungal Pathogens by RAPD
RAPD banding patterns generated using the 10 base primer, AACGCGCAAC, on genomic DNA of
five closely related strains of the pathogen Botrytis cinerea (lanes 1–5), three other strains from the
genus Botrytis (lanes 6, 7 & 8) and several less related harmless fungi Alternaria (9), Aspergillus
(10), Cladosporium (11), Epicoccum (12), Fusarium (13), Hainesia (14), Penicillium (15),
Rhizoctonia (16 & 17), and the host plant, strawberry (18). Lane 0: negative control (no DNA). Lane
M: molecular mass marker. From: Rigotti et al., FEMS Microbiology Letters (2002) 209:169–174.
Reverse Transcriptase PCR
complementary DNA (cDNA) Version of a gene that lacks the introns and is made from the corresponding mRNA by using reverse transcriptase.
reverse transcriptase PCR (RT-PCR) Variant of PCR that allows genes to be amplified and cloned as intron-free DNA copies by starting with mRNA and using reverse transcriptase.
Carrying out RT-PCR on an organism under different growth conditions reveals when the gene under scrutiny was switched on. This allows analysis of which environmental factors bring about expression of any chosen gene.
Reverse Transcriptase PCR
RT-PCR for Gene Expression
DNA Sequencing
Resolved bands in sequencing gel after electrophoresis and autoradiography
Chromatogram of autosequencing
Rapid Amplification of cDNA Ends (RACE)
Rescuing the “lost” ends of cloned genes may be done by a complex PCR based procedure.
The RACE technique generates the complete cDNA in two halves; hence the name rapid amplification of cDNA ends.
The RACE procedure is essentially a modification of RT-PCR, but unique so-called anchor sequences are added to each end of the cDNA to facilitate the PCR portion of the reaction.
Rapid Amplification of cDNA Ends (RACE)
Use of PCR in Medical Diagnosis
Environmental Analysis by PCR
DNA sequences may be amplified by PCR directly from environmental samples.
RT-PCR allows detection of RNA from environmental samples and so reveals whether the target gene is being transcribed.
Genes encoding useful proteins may be cloned from environmental samples without knowing which organism they came from.
It is also possible to isolate useful genes directly by environmental PCR—an approach sometimes referred to as eco-trawling.
Plasmids
Plasmids as Replicons
General Properties of Plasmids
Plasmid Families and Incompatibility
Occasional Plasmids are Linear or Made of RNA
Plasmid DNA Replicates by Two Alternative Methods
Plasmid Addiction and Host Killing Functions
Many Plasmids Help their Host Cells
Antibiotic Resistance Plasmids
Ti-Plasmids are Transferred from Bacteria to Plants
The 2 Micron Plasmid of Yeast
Certain DNA Molecules may Behave as Viruses or Plasmids
Plasmids
Plasmid:
Self-replicating genetic elements that are sometimes found in both prokaryotic and eukaryotic cells. They are not chromosomes nor part of the host cell’s permanent genome. Most plasmids are circular molecules of double stranded DNA although rare linear plasmids and RNA plasmids are known.
Replicon:
Molecule of DNA or RNA that contains an origin of replication and can self-replicate.
General Properties of Plasmids
Most plasmids are circular, made of DNA, and much smaller than chromosomes.
Some plasmids are present in one or two copies per cell whereas others occur in multiple copies.
Some plasmids can transfer themselves between bacterial cells and a few can also transfer chromosomal genes.
Plasmid Families and Incompatibility
Incompatibility: The inability of two plasmids of the same family to co-exist in the same host cell.
Transferability: Ability of a plasmid to move itself from one host cell to another.
Mobilizability: Ability of a non-transferable plasmid to be moved from one host cell to another by a transferable plasmid.
Plasmid Incompatibility
Occasional Plasmids are Linear or Made of RNA
Linear plasmids have special structures to protect the ends of the DNA.
The best-characterized linear plasmids are found in those few bacteria such as Borrelia and Streptomyces that also contain linear chromosomes.
RNA plasmids may have evolved from RNA viruses that have taken up permanent residence after losing the ability to move from cell to cell as virus particles.
End Structures of Linear Plasmids
Plasmid DNA Replicates by Two Alternative Methods
Most plasmids undergo bi-directional replication like bacterial chromosomes.
Some plasmids and many viruses use the rolling circle mechanism for replication.
Transferable plasmids use the rolling circle mechanism during transfer but bi-directional replication when dividing in step with the host cell.
Bi- Directional Plasmid Replication
Rolling Circle Replication
rolling circle replication: Mechanism of replicating double stranded circular DNA that starts by nicking and unrolling one strand and using the other, still circular, strand as a template for DNA synthesis. Used by some plasmids and viruses
Plasmid Addiction and Host Killing Functions
Large plasmids often make toxins that kill the host cell if, and only if, it loses the plasmid DNA.
Many Plasmids Help their Host Cells
Many plasmids carry genes that are beneficial to their host cells, but only under certain environmental conditions.
Antibiotic Resistance Plasmids
R-plasmid or R-factor: Plasmid that carries genes for antibiotic resistance.
ColE plasmid: Small multicopy plasmid that carries genes for colicins of the E group. Used as the basis of many widely used cloning vectors.
Ti-Plasmids are Transferred from Bacteria to Plants
crown gall: Type of tumor formed on plants due to infection by Agrobacterium carrying a Ti-plasmid.
Ti-plasmid: Tumor-inducing plasmid. Plasmid that is carried by soil bacteria of the Agrobacterium group and confers the ability to infect plants and produce tumors.
Only part of the Ti-plasmid enters the plant cell, where it integrates into the plant chromosomes.
Structure of the Ti-Plasmid
Agrobacterium are attracted to an injured region of a plant by sensing molecules of acetosyrigone
Crown Gall Tumor Caused by Agrobacterium
Expression of Genes on T-DNA
The 2-Micron Plasmid of Yeast
2μ plasmid (or 2 μ circle): A multicopy plasmid found in the yeast, Saccharomyces cerevisiae, whose derivatives are widely used as vectors.
Flp recombinase (or flippase): Enzyme encoded by the 2m plasmid of yeast that catalyzes recombination between inverted repeats (FRT sites)
The 2μ Plasmid of Yeast
Selection of transformants using a pUC plasmid vector
Genetic map of bacteriophage λ and λgt11 vector
Bioprocess/fermentation Technology
Sources of carbohydrate and nitrogen for industrial media
Downstream processing
The technology of enzyme production
There is normally a high specific activity per unit dry weight of product.
Seasonal fluctuations of raw materials and possible shortages due to climatic change or political upheavals do not occur.
In microbes, a wide spectrum of enzyme characteristics, such as pH range and high temperature resistance, is available for selection.
Industrial genetics has greatly increased the possibilities for optimising enzyme yield and type through strain selection, mutation, induction and selection of growth conditions and, more recently, by using the innovative powers of gene transfer technology and protein engineering.
Enzyme technology
Approximate annual world production of some industrial enzymes
Industrial applications of enzymes
Genetic engineering and protein engineering of enzymes
Techniques of enzyme / cell immobilization
Some industrial applications of immobilised enzymes
The advantages of immobilized biocatalysts
Limitations of immobilized enzyme techniques
Biological fuel generation
Options for the conversion of biomass to energy
Ethanol from biomass
Potential raw materials for ethanol-fuel production
The gross energy requirements of ethanol produced from different substrates by microbial fermentation
Flow diagram for the production of ethanol
Biodiesel
Biodiesel is obtained from rapeseed as the result of a reaction between the oil and methanol in the presence of a catalyst such as sodium hydroxide at 50◦C, producing an ester and glycerol.
The purified biodiesel has physical and chemical properties that are similar to those of diesel fuel and heating fuel oil. The use of biodiesel does not require any specific engine modifications.
Definition of “Biodiesel” Biodiesel – a fuel comprised of mono-alkyl esters of long chain fatty acids derived from vegetable oils or animal fats, designated B 100. Biodiesel must meet the specifications of ASTM D 6751
What are the main advantages of biodiesel?
Firstly, the energy yielded is considerably greater than that consumed during its production and will increase with improved (genetically engineered) cultivars of rapeseed.
Secondly, it is non-toxic and more than 98% biodegradable, and its contribution to the greenhouse effect is three to five times less than that of diesel.
Above all, it is renewable.
Methane from biomass
Methane gas also exists in the atmosphere and is mainly derived from microbial action in natural wetlands, rice paddies and enteric fermentation in animals, contributing about 20%, 20% and 15% respectively to the total methane flux.
Domestic cattle are the major contributors, producing about 75% of all animal emissions, whereas humans produce about 0.4%. After carbon dioxide, methane is considered to be the next most important greenhouse gas and is expected to contribute 18% of future warming.
The microbiology of methane generation
There are several possible ways by which methane can be produced in a planned economy: from sewage, from agricultural and urban wastes, and in biogas reactors.
In recent years, methanogenesis of the abundantly available agricultural and urban wastes has appeared as an obvious and profitable way to generate energy.
Using urban wastes it should be possible to convert 30–50% of the combustible energy to methane, while with the use of certain other vegetable materials or forages it may be possible to achieve 70% conversion.
Inherent problems that must be overcome
The cost of collection of organic matter only for the purpose of methanogenesis is too expensive;
The rate of methane production is inconsistent and low in most processes, and much research needs to be carried out on the balance of nutrients for process optimizations.
The major problem is the presence of lignin in most agricultural and urban wastes.
When methane is produced by the fermentation of animal dung, the gaseous products are usually referred to as ‘biogas’ and the installations as ‘biogas plants’ or ‘bioreactors’.
Biogas is a flammable mixture of 50–80% methane, 15–45% carbon dioxide, 5% water and some trace gases.
Biogas is produced via biomethanation and is in fact a self-regulating symbiotic microbial process operating under anaerobic conditions, and functions best at temperatures of about 30◦C (The Gobar system).
Under ideal conditions, 10 kg dry organic matter can produce 3 m3 of biogas, which will provide 3 h cooking, 3 h lighting or 24 h refrigeration with suitable equipment.
Economic arguments against large-scale methane production by microbial processes