Ch.09  Biotechnology: Principles and Processes

BIOTECHNOLOGY

​Old biotechnology​

• Microorganisms produced some organic compounds like citric acid, antibiotics etc.

• Types of products were not changed, as they were obtained from natural strains/cell lines

• Only natural capabilities of organisms were exploited

Modern biotechnology

​Human insulin also made from transgenic bacteria, called transgenic proteins

Production technology based on genetic engineering are termed as modern biotechnology, developed during 1970

Definition

• By European Federation of Biotechnology (EFB), biotechnology is the integrated use of biochemistry, microbiology and engineering sciences in order to achieve technological (industrial) application of capabilities of microorganisms, cultured tissues/cells and parts thereof

PRINCIPLES OF BIOTECHNOLOGY​

Two main techniques of modern biotechnology

Genetic engineering

• Technique to alter nature of genetic material (DNA, RNA) to introduce these into host organisms and thus change phenotype

Chemical engineering

• Involves maintenance of sterile microbial contamination free condition in chemical engineering processes to have growth of only the desired microorganism/eukaryotic cell in large quantities for the manufacture of biotechnological products such as antibiotics, vaccines, enzymes, medicines, hormones etc.

Conceptual development of principles of genetic engineering​

• Genetic engineering is a kind of biotechnology which deals with manipulation of genetic material in vitro

• Genetic engineering is based on 2 main discoveries

  • Presence of plasmids in Bacteria, can undergo replication along with or independent of chromosomal DNA

  • Restriction endonucleases which can break DNA at specific sites, called molecular scissors

Role of Paulberg​

• Introduced gene of SV-40 virus into bacterium with ʎ phage

• Got Nobel 1980

• Father of genetic engineering

Technique of genetic engineering includes

• Formation of recombinant DNA (rDNA)

• Use of gene cloning

• Gene transfer, introducing only desirable gene

Construction of first artificial recombinant DNA Molecule​

• Stanley Cohen 1972, Herbert Boyr isolated antibiotic resistant gene from plasmid of Salmonella and cloned in E. coli

• 3 basic steps in creating GMO or transgenic organism

  • Identification of DNA with desirable gene

  • Introduction of identified DNA into the host

  • Maintenance of introduced DNA into host and transfer of DNA to its progeny

TOOLS OF RECOMBINANT DNA TECHNOLOGY​

ENZYMES​

Lysing enzymes

• Lysozyme to open bacteria

• Cellulase for plant cells

• Chitinase for fungi​

Cleaving enzyme

• Exonuclease, remove from terminal ends 5’ or 3’

• Endonuclease, cuts at specific position within DNA, do not cleave ends and involve only one strand of DNA duplex

• Restriction endonuclease, cut duplex at specific points, single stranded free ends are sticky which can be joined by ligase

Synthesizing enzymes

• Reverse transcriptase, synthesize cDNA from on RNA template

• DNA polymerase, synthesise of complementary DNA Strands on DNA templates

Joining enzymes

• ​T4 Ligase

Alkaline phosphatase

• cut phosphate group at 5’ end so no circularisation

RESTRICTION ENDONUCLEASE​

• Isolated first by W Arber in 1962 in bacteria (Arber and Nathan Nobel in 1978)

• Found in bacteria, 2 enzymes, one adding methyl group to DNA and another cutting enzyme called RE discovered by S. Linn & W ArberBacteria has restriction and modification system for protection from bacteriophages

Type I​

• Consists of 3 different subunits

• Require ATP, Mg++, and S-adenosyl methionine for restriction

• Restriction and methylation

• 15 bps recognised, cut 1000 pb away from 5’ end

• Not used

Type II

• Simple and require Mg++ ions, Used in vitro

• Recognise 4-8 bp

• Cut within

• More than 350 such enzyme now isolated

• Are used in genetic engineering

• No ATP needed for cleaving

• It makes cleavage in both strands

Type III

• Consists of 2 subunits

• Require ATP, Mg++, S-adenosyl methionine for restriction

• Intermediate properties between type I and II

• t Does both restriction and methylation

• Not used

• H.O. Smith, K W Wilax, T J Kelly 1968, discovered 1st enzyme Hind II. Now 900 known enzymes

LIGASE​

• Most often used is T4 DNA Ligase (discovered by HG Khurana in 1969)

ALKALINE PHOSPHATASE (AP)

• Removes a phosphate group from 5’ end of double strand or single stranded DNA or RNA, can’t circularize

SYNTHESIZING ENZYMES

REVERSE TRANSCRIPTASE

• Synthesize DNA or complimentary DNA (cDNA) by using mRNA as template, discovered by Temin and Baltimore in 1970

DNA POLYMERASE

• Helps in DNA synthesis on DNA Template or complementary DNA, discovered by A. Kornberg and co-workers in E.coli in 1956

NOMENCLATURE

EcoRY13 as Eco RI

FUNCTIONING OF RE

• In bacteria they function as defence system called Restriction and Modification System, first explained by Wemer Arber 1965

• Inspect and cut, by restriction endonuclease

• Modification enzyme adds a methyl group to one or two bases usually “within” the sequence reorganized by restriction enzyme

• Once a base in a DNA sequence is modified by addition of a methyl group, restriction enzyme fails to recognize and couldn’t cut that DNA. This is how bacteria modify and protect its DNA from cleavage by RE

• First RE was Hind II

PALINDROMIC Nucleotide Sequence

Special sequences recognised, Of each RE, for

​

5’-----GAATTC-------3’

3’-----CTTAAG-------5’

• Like MADAM, MALAYALAM

• RE cuts strand of DNA, a little away from centre of palindrome site but between same two bases of opposite strands

• This leaves sticky sites

AGAROSE GEL ELECTROPHORESIS​

• Separation and isolation of DNA fragments

• By A. Tselius 1937

• Agarose is a polysaccharide from sea weeds

• Current passed in solution

• They have same electric charge hence, Move according to size towards anode, smaller reaching farther

• Ethidium bromide and seen in UV light as orange bands

• Bands are cut and extracted from gel piece, called elution (removal of adsorbent)

CLONING VECTORS/VEHICLE DNA/CARRIER DNA

PLASMID VECTORS

• Natural in bacteria in some yeast

• Extra chromosomal, self-replicating, usually circular, double stranded

• Not essential for normal cell growth and division

• Confers some traits to organisms, resistance to antibiotics or toxins

• Present as 1 or 2 copies or in multiple copies (500-700) inside host

• Have been modified to serve as vectors

• Take part in transformation and conjugation

• Discovered By W Hays and J. Lederberg

• PBR 322 1977

• PUC8

  • Better, with Lac z β-galactosidase

  • Insertional inactivation of this enzyme leads to no or white colour (recombinant)​

Features that are required to facilitate cloning into a vector

Origin of replication (ori)

• For initiating replication

• Prokaryotic DNA has a single ori while eukaryotic DNA may have more than one

• Generally, 20-30 copy

• Desirable to attach to vector with high copy number

Selectable marker

• Help in selecting transformants

• Antibiotic resistant gene used in E. coliixation

• Common E.coli cells not resistant against any of these antibiotics

• Gene for production of toxic substance

• For tumour formation

• For N2 F

Cloning site (recognition sites)

• Preferably single

• Rop codes for the proteins involved in replication of plasmid

​

Vectors for cloning genes in plant and animals

Agrobacterium tumefaciens

• A soil inhabiting bacteria of several dicot plants is able to transfer a piece of DNA called TDNA, that causes tumours, called crown galls

• Tumor formation is induced by Ti plasmid

• Bacteria is called natural genetic engineer of plants

• Similarly, retroviruses (cause leukosis or sarcoma type of cancer is animals are able to change normal cells into cancerous cells

• Ti plasmid have been modified into vector which is not pathogenic to plants, still able to deliver genes of interest

• Similarly, retroviruses are used to carry genes into animals

Shuttle vector in both prokaryotes and eukaryotes

Bacteriophage vector

• Inject DNA which integrate as Prophage and multiply they burst out lytic way

ii. Most common are:

iii. Lambda Phage Vector

1. Double stranded, linear DNA genome of 48, 502 bp, in which the 12 bases at each end are unpaired but complementary

2. These ends are therefore sticky and are referred to as cos sites, important for packing DNA into phage head

3. Lambda phage remains linear in phage head, but within E. coli cells two cohesive ends join to form a circular molecule necessary for replication.

4. These vectors allow cloning of DNA fragments up to 23 Kb length (1kb= 1000 nucleotide long bases)

5. Up to 23 kb DNA cloned

6. M13 Phage Vector-filamentous phage

iv. M13 PAHGE

1. It is filamentous phage which infects E.coli having F-pilli

2. Its genome is a single stranded, circular DNA or 6407 bp

3. Foreign DNA can be inserted into it without disrupting any of essential genes

4. After the M13 Phage DNA enters the bacterial cell, it is converted to a double stranded molecule known as replicative form of RF, which replicates until there are 100 copies in cell and single stranded copies of the genome are produced and extruded from the cell as M13 particle

5. The major advantages of developing vectors based on M13 are that its genome is less than 10kb length

6. The RF can be purified and manipulated exactly like a plasmid

7. In addition, genes cloned in M13 based vectors can be obtained in the form of single stranded DNA

v. Why bacteriophage vectors more advantageous than plasmid vectors

1. Can be Used for large DNA

2. Can be easily detected at time of cloning experiment

g. Some other cloning vectors

i. Cosmid

1. ‘cos’ site of lambda + plasmid (MID), cos site is cohesive

2. Can be used to clone DNA fragments up to 45kb in length

ii. Bacterial Artificial Chromosomes (BAC vectors)

1. These are vectors based on natural extrachromosomal plasmid of E. coli

2. These vectors can accommodate up to 300-350kb of DNA and are also being used in genome sequencing project

iii. Yeast Artificial Chromosome (YAC) vectors

1. Used to clone DNA of more than 1 Mb (mega base 106) in size, therefore, they have been exploited extensively in mapping the large genomes, e.g. In Human Genome Project

2. These vectors contain the telomeric sequence, the centromere and autonomously replicating sequence from yeast chromosomes

3. They also contain restriction enzymes sites and genes which act as selectable markers in yeast

iv. Phagemid vector

1. Bacteriophage + plasmid

2. Carry large DNA

v. Animal and Plant viral vectors

1. A vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979

2. Since 1979, a number of vectors based on other types of viruses like Adenovirus and Papilloma virus (benign tumour warts in humans) have been used to clone genes in mammals

3. At present, retroviral vectors are the most commonly used vectors for cloning genes in mammalian cells

4. In case of plants, plant viruses like cauliflower mosaic virus, TMV and Gemini viruses were used but with limited success

vi. Transposons as Vectors

a. Are Unit of DNA which can move from one DNA to other, hence mobile

b. They are also called transposable elements or mobile genes or jumping genes

c. Transposons were first observed by Clintock in maize plants

vii. Shuttle vectors

1. They can exist in both the eukaryotic cell and E. coli.

2. Such vectors contain two types of origin of replication and selectable marker genes, on type that functions in eukaryotic cell and another that functions in E.coli

3. An example of is yeast episomal plasmid Yep.

4. In case of plants a naturally occurring plasmid of Agrobacterium tumefaciens called Ti plasmid has been suitable modified to function as vector

5. Most of eukaryotic vectors are shuttle vectors

viii. Passenger DNA

1. DNA which is transferred from one organism into another by combining it with vehicle DNA. 3 TYPES:

a. cDNA or copy DNA or complementary DNA

i. it is synthesized on RNA template with reverse transcriptase

ii. DNA template is isolated from RNA-DNA complex by using alkaline phosphatase enzyme

iii. A complementary DNA strand is then synthesized on the isolated single stranded DNA template with help of DNA polymerase

iv. cDNA duplex so formed can be joined to vehicle DNA for introduction into host cell

b. synthetic DNA

i. Synthesized with DNA Polymerase on DNA template or from free

Page 15 of 22

deoxyribonucleotide triphosphates, DNA Polymerase and metal ions and a sequence of viral DNA to act as primer

ii. Shows biological activity when introduced in bacteria, this DNA coded for formation of virus particles similar to one from which primer DNA was taken

h. Artificial synthesis of DNA without a template first by HG Khorana in 1965

i. Random DNA, small fragments formed by breaking a chromosomes of an organism with help of restriction endonucleases

E. COMPETENT HOST (for transformation with rDNA)

i. DNA Mediated Gene Transfer (Vector Mediated Gene Transfer)

1. Many host cells, including E. coli, yeast, animal and plant cells, are available for genetic engineering. Kind of host cell depend on aim of cloning experiment

2. For expression of some eukaryotic proteins, eukaryotic cells may be the preferred genes because they offer several advantages

3. Yeast are simplest eukaryotic organisms and like bacteria are single celled, genetically well characterized, easy to grow and manipulate

4. Can be grown in small culture vessels and large-scale bioreactor

5. Plant and animals cells may also be used as hosts in gene manipulation experiments and for protein expression either in tissue culture or as cells in the whole organism to create genetically modified plants and animals

6. Since DNA is hydrophilic molecule, it can not pass through membranes, so bacterial cells must be made capable to take up DNA

7. This is done by treating them with a specific concentration of divalent cation, such as Calcium which increases the efficiency with which DNA enters the bacterium through pores in its cell wall

8. rDNA can then be forced into such cells by incubating cells with recombinant DNA on ice, followed by placing them at 42 C (hear shock), and then putting them back on ice

9. this enables the bacteria to take up Recombinant DNA

10. transfer of DNA into eukaryotic cell is called transfection

ii. Direct or Vector less Gene Transfer

1. It is the process of gene transfer into the host cell without using a vector. This is possible by following 4 important methods

a. Micro Injection

i. Foreign DNA is directly injected into the nucleus of animal cell or plant cell by using micro needles or micro pipettes

ii. It is used in oocytes, eggs and embryo

iii. Alec Jeffreys (1993) of Human Genome Centre, has cured a mice that inherited a neuromuscular disease which is like muscular dystrophy of humans

b. Electroporation

i. Formation of temporary pores in plasma membrane of host cells by using lysozyme or calcium chloride

ii. These pores are used for introduction of foreign DNA

c. CHEMICAL Mediated Gene Transfer

i. In this method certain chemicals such as polyethylene glycol (PEG) help foreign DNA to enter host cell

d. Biolistic Method/Gene Gun Method

i. Introducing DNA into cells that involves bombardment of cells with high velocity microprojectiles coated with DNA

ii. In biolistic method tungsten or gold particles, coated with foreign DNA are bombarded into target cells at very high velocity

iii. Suitable for plants yet this also used in animals

F. PROCESS OF rDNA TECHNOLOGY

i. Isolation of Genetic Material, DNA

1. Cells have to be break open by and purified

2. Enzymes are lysozyme, cellulase, chitinase

3. Histones, RNA are removed by treating with appropriate enzymes

4. Purified DNA finally precipitates out after the addition of chilled ethanol, like fine threads, called spooling (spool = reel)

ii. Cutting of DNA at specific locations

1. By restriction enzymes

2. gel electrophoresis is used to check progression of digestion

3. this is also repeated with vector

4. Formation of rDNA

a. After cutting source DNA and vector DNA with a specific restriction enzyme, the cut-out gene of interest and vector are mixed and ligase enzyme is added

b. This forms rDNA or hybrid DNA or Chimeric DNA

iii. Amplification of gene of interest, using PCR

1. Invented byy Kary Mullis in 1985

2. This is DNA replication in vitro

3. It results in selective amplification of a specific region of a DNA molecule and so can also be used to generate a DNA fragment for cloning:

a. DNA template

b. Two nucleotide primers

i. Are oligo-nucleotides, that hybridize to target DNA region, one to each strand of double helix are required

ii. These primers are oriented with therir ends facing each other allowing synthesis of DNA towards one another

c. Enzyme-Taq polymerase

4. Working Mechanism of PCR

a. Denaturation

i. 94⁰C, each strand then act as template

b. Annealing

i. 40-60⁰C, of oligo nucleotide primers anneal (hybridize) to each of single stranded template DNA since the sequence of primers is complementary to 3’ ends of template DNA

c. Extension (polymerization)

i. 72⁰C, Taq DNA polymerase synthesizes the DNA region between the primers, using DNTPs and Mg2+

ii. it means primers are extended towards each other

5. Application of PCR

a. Detection of pathogens

i. Detect HIV

b. Diagnosis of specific mutations

i. Phenylketonuria, muscular dystrophy, sickle cell anaemia, hepatitis, chlamydia and tuberculosis can be diagnosed

c. DNA finger printing

d. Detection of specific microorganisms

e. In prenatal diagnosis

i. Genetic diseases in foetus before birth

f. Diagnosis of plant pathogens

g. In palaeontology

i. Used to clone DNA fragments from mummified remains of humans and extinct animals like wooly mammoth and dinosaurs

h. In gene therapy

i. Immense help in monitoring a gene in gene therapy experiments

iv. Preparation and insertion of recombinant DNA int the host cell/organisms

1. Vector DNA and foreign DNA carrying gene of interest are cut by same restriction enzymes for sticky sites and ligated forming recombinant DNA

2. Both direct and indirect methods are used to introduce ligated DNA into host cells

3. It is selected by selectable markers

G. OBTAINING THE FOREIGN GENE PRODUCTS

i. Desirable proteins are obtained from expressed DNA

ii. One has to maintain optimum conditions to induce expression

iii. On large scale done

iv. Bioreactors (fermenters)

1. Provide optimal conditions as temperature, pH, substrate, vitamins, oxygen, salts

2. Batch culture

a. Nutrients and microorganisms are put in a closed reactor and not

3. Continuous culture

v. Types of bioreactors

1. Simple stirred tank bioreactor and Sparged stirred tank bioreactor

a. Sterile air bubbles are sparged. The surface area for oxygen transfer is increased

b. Raw material as glucose is fermented to alcohol etc

c. A bioreactor has oxygen delivery system, a foam control system, temperature and Ph control system and a sampling port

d. Term fermentation is also used here for all processes, aerobic and anaerobic

e. All operations are carried out under sterile conditions to avoid contamination of the culture

f. Product is either the cells themselves or some useful cell products

g. 2 basic types of fermentation are batch and continuous fermentation nutrients and microorganisms are put in a closed reactor and not changed from outside once fermentation starts

h. Continuous culture maintain cells in their physiologically most active lag, exponential phase

i. When nutrients are utilized, the products is separated from microorganisms

j. In continuous fermentation nutrients are replaced as fast as they are used and products are removed as soon as they are made

k. Stirred tank bioreactor are well suited for large scale production of microorganisms under aseptic conditions for a number of days

l. Drawback is that it is expensive to run it

H. DOWNSTREAM PROCESSING

i. Products undergoes through some processes before finished product for marketing

ii. Includes separation, purification etc. collectively called downstream processing

iii. Product subjected to quality control testing and kept in suitable preservatives

iv. If drugs then undergo clinical trials

Different for different products