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1.3D: Modern Microbiology - Biology

1.3D: Modern Microbiology - Biology


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Learning Objectives

  • Discuss the fundamental aspects of microbiology

Modern microbiolgy began with the discovery of microbes, and the scope and scale of the field continues to expand today. While there is some debate, modern microbiology is accepted by most to begin with observations by the Dutch draper and haberdasher, Antonie van Leeuwenhoek, who lived for most of his life in Delft, Holland. In 1676, van Leeuwenhoek observed bacteria and other microorganisms, using a single-lens microscope of his own design. While van Leeuwenhoek is often cited as the first to observe microbes, Robert Hooke made the first recorded microscopic observation, of the fruiting bodies of molds, in 1665.

It has been suggested that a Jesuit priest called Athanasius Kircher was the first to observe microorganisms. One of his books contains a chapter in Latin, which reads in translation – “Concerning the wonderful structure of things in nature, investigated by Microscope.” Here, he wrote “who would believe that vinegar and milk abound with an innumerable multitude of worms. ” He noted that putrid material is full of innumerable creeping animalcule. These observations antedate Robert Hooke’s Micrographia by nearly 20 years and were published some 29 years before van Leeuwenhoek saw protozoa.

The field of bacteriology (later a subdiscipline of microbiology) was founded in the 19th century by Ferdinand Cohn, a botanist whose studies on algae and photosynthetic bacteria led him to describe several bacteria including Bacillus and Beggiatoa. Cohn was also the first to formulate a scheme for the taxonomic classification of bacteria and discover spores. Louis Pasteur and Robert Koch were contemporaries of Cohn’s and are often considered to be the father of microbiology and medical microbiology, respectively. Pasteur is most famous for his series of experiments designed to disprove the then widely held theory of spontaneous generation, thereby solidifying microbiology’s identity as a biological science. Pasteur also designed methods for food preservation (pasteurization) and vaccines against several diseases such as anthrax, fowl cholera, and rabies.

Koch is best known for his contributions to the germ theory of disease, proving that specific diseases were caused by specific pathogenic microorganisms. He developed a series of criteria that have become known as the Koch’s postulates. Koch was one of the first scientists to focus on the isolation of bacteria in pure culture resulting in his description of several novel bacteria including Mycobacterium tuberculosis, the causative agent of tuberculosis. While Pasteur and Koch are often considered the founders of microbiology, their work did not accurately reflect the true diversity of the microbial world because of their exclusive focus on microorganisms having direct medical relevance.

It was not until the late 19th century and the work of Martinus Beijerinck and Sergei Winogradsky, the founders of general microbiology (an older term encompassing aspects of microbial physiology, diversity, and ecology), that the true breadth of microbiology was revealed. Beijerinck made two major contributions to microbiology: the discovery of viruses and the development of enrichment culture techniques. While his work on the tobacco mosaic virus (TMV) established the basic principles of virology, it was his development of enrichment culturing that had the most immediate impact on microbiology by allowing for the cultivation of a wide range of microbes with wildly different physiologies. Winogradsky was the first to develop the concept of chemoautotrophy and to thereby reveal the essential role microorganisms played in geochemical processes. Specifically, he was responsible for the first isolation and description of both nitrifying and nitrogen-fixing bacteria.

Key Points

  • There is some debate as to who was exactly first, but Antonie van Leeuwenhoek, Athanasius Kircher, and Robert Hooke were the first people to view microbes using some of the first self-built microscopes.
  • Ferdinand Cohn, Louis Pasteur, and Robert Koch were pioneers in bacteriology, the discovery and understanding of the subset of microbes that are bacteria. This had a direct and immediate impact on food storage and disease causality.
  • Martinus Beijerinck and Sergei Winogradsky are credited with the discovery of general microbiology, which laid the ground work for our understanding of microbial physiology, diversity, and ecology.

Key Terms

  • chemoautotrophy: When a simple organism, such as a protozoan, derives its energy from chemical processes rather than photosynthesis.
  • pasteurization: heat-treatment of a perishable food to destroy heat-sensitive vegetative cells followed by immediate cooling to limit growth of the surviving cells and germination of spores
  • rabies: a viral disease that causes acute encephalitis in warm-blooded animals and people, characterised by abnormal behaviour such as excitement, aggressiveness, and dementia, followed by paralysis and death
  • animalcule: An older term for a minute or microscopic animal or protozoan.

The Modern Journal of Microbial Biology (MJMB) is an open-access peer-reviewed journal committed to publishing high-quality articles in the field of Microbial Biology from across the globe. The journal is owned by Prof. Dr. Emad Abd Elmoniem Mohamed Ebada and published by the Scientific Scholar. The journal aims to present the most current information in Microbial Biology that is relevant to the current practice.

Focus & Scope

The Modern Journal of Microbial Biology covers topics related to Microbiology, but are not limited to Agricultural Microbiology, Bacteriology, Evolutionary Microbiology, Food Microbiology, Industrial Microbiology, Medical Microbiology, Microbial Biology, Microbial Biotechnology, Microbial Ecology, Microbial Genetics, Pharmaceuticals Microbiology, Plant-Microbe interactions, Systematic Microbiology, Veterinary Microbiology, Virology.

Aims to publish the most complete and reliable information on the discoveries, experimental approaches and current research works on Microbial ecology, Medical Microbiology, Microbial physiology, etc., with strict ethical policies and standards to ensure high quality of scientific works related to the field.

Audience

Our audience includes all scientists in the field of microbiology (including but not limited to Microbiologists, Medical Researchers, Geneticists, Veterinary science professionals, Pharmaceutical Researchers), Graduated students, Governmental and private organizations, and Public.

Review process

MJMB has a highly rigorous double-blinded peer-review process that makes sure that manuscripts are scientifically accurate, relevant, novel and important. Authors disclose all conflicts of interest, affiliations and financial disclosures such that the published content is not biased.

In-house submissions that contain the work of any editorial board member, are not allowed to be reviewed by that editorial board member and all decisions regarding this manuscript are made by an independent editor. In addition, these manuscripts are reviewed by the two external reviewers. This is also disclosed in the published manuscript under the section of Conflict of Interest.

Editorial team

The editorial board comprises of a panel of international experts in Microbial Biology from various countries to provide their expertise and guidance to the Editor-In-Chief in maintaining the clarity of content and focus of the journal’s purpose.

About the Editor

Prof. Dr. Emad A. Abada PhD

Prof. of Molecular Biology and Microbiology at Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, Egypt. He has got his B.Sc. in Botany from the faculty of Science, Cairo University, Egypt, 1992. He has got his master’s in Microbiology from the Faculty of Science, Cairo University, Egypt, 1996. He has got his PhD. from the Institute of Microbiology and Molecular Biology, Faculty of Science, Giessen University, Giessen, Germany, 2002. He was awarded a Post-doc fellowship at the faculty of Science, Chonnam University, South Korea from 2006-2008. According to googol scholar, Prof. Dr. Abada had published 33 papers including 2 book chapters in the field of Microbiology, Biotechnology and Molecular Biology with 281 citations and 10 h-index, mostly in impact factor international journal. Prof. Abada was the supervisor of many master and doctor students. Prof. Abada was a principal investigator and co-investigator of many research projects funded by high institution funder. He participated in many international and local conferences during his carrier.

Facts about the Journal

MJMB is a peer-reviewed international journal with a double-blind review process. Its frequency of publication is continuous. The journal is published in E-Journal format and the journal is also available in print on demand.

Benefits

A manuscript published in the journal can be read and downloaded by readers all over the world without any charges or fees. The author retains the copyright of the manuscript and the figures. This allows authors to extend their exposure in the world of medical research and get worldwide recognition for their work.

Abstracting and Indexing Information

The journal is registered with the following abstracting partners:

Google Scholar, CrossRef, ReadCube, Scilit

Journal Ethics

The journal is committed to meeting and upholding ethical guidelines at all stages of the publication process. We follow the industry associations, such as the Committee on Publication Ethics (COPE), International Committee of Medical Journal Editors (ICMJE), and World Association of Medical Editors (WAME), that set standards and provide guidelines for best practices to meet these requirements. For a summary of our specific policies regarding duplicate publication, conflicts of interest, patient consent, etc., please visit our Ethical Guidelines Section.

Plagiarism Policy

We provide plagiarism check software (iThenticate) on the Editor’s and Reviewer’s panel in the manuscript management system. We encourage our editors and reviewers to use the plagiarism check. The manuscript’s found to have plagiarism is rejected.

Archival Policy

We use PORTICO for our third-party repository for all articles published with us. All our manuscripts are assigned a DOI number from CROSSREF, and we are members of Crossref https://crossref.org.

Open Access Publication and Creative Commons Licensing

MJMB is an open-access journal, and manuscripts published are distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

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Articles published online under the Ahead of Print model are considered published and can be cited and quoted using the DOI as the reference source. The journal has a policy that changes will not be made after publication of an article without following accepted procedures for making corrections to the scientific record.

Advertisements

While advertisements are crucial to this journal to be able to keep all content free for everyone, ethical considerations are in place (https://scientificscholar.com/adv-policies/) to ensure the integrity of the journal and its content are not biased or influenced by the advertisement.

“Pop-up” and “Banner” advertisement appears at a fixed location on the page and does not interfere with the reading/display of the manuscript in any form. (https://modernjmicrobiol.com/advertise/)

All advertisements are approved in consultation with Editor-In-Chief/the Editorial Board.

Advertisers submit their advertisements without prior information of the journal content to be published.

Subscription Information

MJMB is a continuous publication and published manuscripts in the journal can be read and downloaded by readers all over the world without any charges or fees.


Branches of Microbiology

By Taxonomy

  • Bacteriology: the study of bacteria.
  • Immunology: the study of the immune system. It looks at the relationships between pathogens such as bacteria and viruses and their hosts.
  • Mycology: the study of fungi, such as yeasts and molds.
  • Nematology: the study of nematodes (roundworms).
  • Parasitology: the study of parasites. Not all parasites are microorganisms, but many are. Protozoa and bacteria can be parasitic the study of bacterial parasites is usually categorized as part of bacteriology.
  • Phycology: the study of algae.
  • Protozoology: the study of protozoa, single-celled organisms like amoebae.
  • Virology: the study of viruses.

By Type of Research

Microbiology research, like other fields of scientific research, can be subdivided into the categories of pure and applied. Pure (or basic) research is exploratory and conducted in order to better understand a scientific phenomenon, while applied research is based on information gleaned from pure research and used to answer specific questions or solve problems.
Pure microbiology research includes:

  • Astromicrobiology: the study of the origin of life on Earth, and the search for extraterrestrial life.
  • Evolutionary microbiology: the evolution of microorganisms.
  • Cellular microbiology: the study of the structure and function of microbial cells.
  • Microbial ecology
  • Microbial genetics
  • Microbial physiology
  • Systems microbiology: mathematical/computational modeling of the activities of microbiological systems.

While applied microbiology research includes:

  • Agricultural microbiology: the study of microorganisms that interact with plants and soils.
  • Food microbiology: the study of microorganisms that spoil food or cause foodborne illnesses. Can also study how microorganisms are used in food production, such as fermentation of beer.
  • Medical microbiology: the study of microorganisms responsible for human disease.
  • Microbial biotechnology: using microbes in industrial or consumer products.
  • Pharmaceutical microbiology: the study of microorganisms used in pharmaceutical products, such as vaccines and antibiotics.


This is an image of bacteria colonies growing on an agar plate.


Biotechnology: Useful Notes on Traditional and Modern Biotechnology

Biotechnology deals with techniques of using live micro-organisms, plant or animal cells or their components or enzymes from organisms to produce products and processes (ser­vices) useful to human beings.

The term biotechnology was coined in 1917 by a Hungarian Engineer, Karl Ereky to describe a process for large scale production of pigs.

Gene manipulation is a fast emerging science. It started with the development of recom­binant DNA molecules. It is named variously as DNA manipulation biotechnology, recom­binant DNA technology and genetic engineering.

The technology mostly involves cutting and pasting of desired DNA fragments. It is based on two important discoveries in bacteria:

(i) Presence of plasmids in bacteria which can undergo replication along with and indepen­dent of chromosomal DNA.

(ii) Restriction endonucleases (Arber, Nathan and Smith 1970 Nobel Prize in 1978) which can break DNA at specific sites.

They are appropriately called molecular scissors. Berg (1972) was able to introduce a gene of SV-40 into a bacterium with the help of lambda phage. Berg is often considered “father of genetic engineering”. He was awarded Nobel Prize in 1980. The science of recombinant technology took birth when Cohen and Boyer (1973) were able to introduce a piece of gene containing foreign DNA into plasmid of Escherichia coli.

Old Biotechnology (Traditional Biotechnology):

Microorganisms were first used to produce some organic compounds like citric acid. They were also used to produce antibodies. The levels of production of penicillin yield has been improved But the types of products are not changed. They remain the same as those obtained from the natural strains/cell lines. In all these processes, only the natural capabilities of the organisms and cells are exploited. These activities are called old biotechnology.

Modern Biotechnology:

Human insulin is also produced from a transgenic Escherichia coli stain that contains and expresses the insulin gene. Proteins produced by transgenes are called recombinant proteins. The production technologies based on genetic engineering are termed as modern biotechnology. It developed during 1970.

Definition of Biotechnology:

A definition of biotechnology which covers both traditional views and modern molecular biotechnology has been given by European Federation of Biotechnology (EFB). According to EFB. “Biotechnology is the integrated use of biochemistry, microbiology and engineering sciences in order to achieve technological (industrial) application of the capabilities of microorganisms, cultured tissues/cells and parts thereof’. Thus definition of biotechnology involves two common factors. First the use of biologi­cal agents and second the product or service is generated for the well being of humans.`

I. Principles of Biotechnology:

Two Main Techniques of Modern Biotechnology:

The two main techniques that gave birth to modem biotechnology are as follows:

(a) Genetic Engineering:

It includes techniques to alter the nature of genetic material (DNA and RNA) to introduce these into host organisms and thus change the phenotype of the host organism.

(b) Chemical Engineering:

It 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.

1. Conceptual Development of the Principles of Genetic Engineering:

Genetic engineering is a kind of biotechnology which deals with the manipulation of genetic material by man in vitro.

Genetic engineering is based on two important discoveries in bacteria.

(i) Presence of plasmids in bacteria which can undergo replication along with and independent of chromosomal DNA.

(ii) Restriction endonucleases (Arber, Nathan and Smith 1970 Nobel Prize in 1978) which can break DNA at specific sites. They are appropriately called molecular scissors or biological scissors.

In 1972 genetic engineering was started by Paul Berg. Berg (1972) was able to introduce a gene of SV-40 virus into a bacterium with the help of lambda phage. Berg is often considered “father of genetic engineering”. He was awarded Nobel Prize in 1980.

The technique of genetic engineering includes:

(i) Formation of ‘recombinant DNA’ (rDNA).

(iii) Gene transfer. It permits to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.

A piece of DNA which is introduced into the alien (foreign) organism would not be able to multiply itself in the organism but when it gets incorporated into the genetic material of the recipient, it may multiply and be inherited along with the host DNA, because the alien piece of DNA has become part of chromosome which possesses the ability to replicate.

There is a specific DNA sequence called the origin of replication in a chromosome that is respon­sible for initiating replication. Thus, an alien DNA linked with the origin of replication can replicate and multiply itself in the host organism. It is also called the cloning, i.e., forming multiple identical copies of any template DNA.

2. Construction of the First Artificial Recombinant DNA Molecule:

The first recombinant DNA was constructed by Stanley Cohen and Herbert Boyer in 1972. They cut the piece of DNA from a plasmid carrying antibiotic resistance gene in the bacterium Salmonella typhimurium. Cutting of a piece of DNA from a plasmid was with the help of restriction enzymes (also called molecular scissors or chemical scalpels).

The piece of foreign DNA cut from the plasmid was linked with the plasmid DNA acting as vector. Linking of the piece of foreign DNA with vector was done with the help of the enzyme DNA ligase which acts on cut DNA molecules and join their ends. This newly formed DNA having integrated fragment of antibiotic resistant gene is called recombinant DNA.

The vectors is used to transfer, recombinant DNA to E. coli. This transfer of recom­binant DNA is similar to the transfer of malaria parasite from diseased person into the healthy person through female Anopheles mosquito (acts as an insect vector).

When this recombi­nant DNA is transferred into Escherichia coli, it could replicate in the new host cell in the presence of DNA polymerase enzyme and make multiple copies of recombinant DNA. The ability to multiply copies of antibiotic resistance gene in E. coli was termed as cloning of antibiotic resistance gene in E. coli.

Three basic steps in creating genetically modified organism (GMO) or transgenic organism. GMO contains a foreign gene/segment of DNA. These three basic steps are as follows.

(i) Identification of DNA with desirable genes.

(ii) Introduction of the identified DNA into the host.

(iii) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

II. Tools of Recombinant DNA Technology:

Three types of “biological tools” are used in the formation of recombinant DNA. These are as follows:

(B) Cloning Vectors (Vehicle DNA)

(C) Competent host (for transformation with recombinant DNA)

These enzymes are used to open up the cells to get DNA for genetic experiments. Lysozyme is usually used to dissolve the bacterial cell wall. In plant cell, cell wall is made up of cellulose, while in fungi, it is made up of chitinase.

These enzymes are used to break DNA molecules. They are of three types— exo-nucleases, endonucleases and restriction endonucleases.

They remove nucle­otides from the terminal ends (either 5′ or 3′) of DNA in one strand of duplex.

They make cuts at specific position within the DNA. These enzymes do not cleave the ends and involve only one strand of the DNA duplex.

(C) Restriction endonucleases:

They cut DNA duplex at specific points. Their single stranded free ends are called ‘sticky ends’ (Fig. 11.3) which can be joined end to end by DNA ligases.

Restriction Endonucleases — The Molecular Scissors:

Restriction endonuclease was isolated for the first time by W. Arber in 1962 in bacteria. They are called “molecular scissors or biological scissors”. In 1978 Arber, Smith and Nathan were awarded the Nobel Prize for the discovery of restriction endonuclease.

They recognize the base sequence at palindrome sites in DNA duplex and cut its strands. For example, restriction endonuclease EcoR 1 found in the colon bacteria E. coli, recognizes the base sequence GAATTC in DNA duplex and cuts its strands between G and A as shown below : Only restriction enzymes type II are used in gene manipulation for two reasons,

(a) No ATP is needed for the cleaving action,

(b) It makes cleavage or cut in both the strands of DNA molecule.

(1) Types of Restriction Endonucleases:

Three main types of restriction endonu­cleases are type I, type II and type III.

Type I Restriction Endonucleases:

These enzymes consist of 3 different subunits. They require ATP, Mg 2+ and S-adenosyl methionine for restriction. Type I restriction endo­nucleases recognize specific sites within the DNA but do not cut these sites. Thus they are not used in recombinant DNA technology.

Type II Restriction Endonucleases:

These enzymes are simple and require Mg 2+ ions for restriction. Out of the three types, only type II restriction enzymes are used in recombinant DNA technology.

Тyре III Restriction Endonucleases:

These enzymes consist of two different subunits. They require ATP, Mg 2+ and S-adenosyl methionine for restriction. They recognize specific sites within DNA but do not cut these sites, therefore, these restriction endonucleases are not used in recombinant DNA technology. They have intermediate properties between type I and type II.

Differences Between Тyре I, Тyре II and Type III Restriction Endonucleases:

Type I Type II Тyре III
1. Enzyme structure consists of 3 different subunits. Enzyme structure is simple. Enzyme structure consits of two different sub units.
2. They require ATP, Mg 2+ S-adenosyl-methionine for restriction. They require Mg 2+ ions for restriction. They require ATR Mg 2+ and S-adenosyl, methionine for restriction.
3. They recognize specific sites within DNA but do not cut these sites. They recognize specific sites within the DNA and cut these sites. They recognize specific sites within the DNA but do not cut these sites.
4. They are not used in recombinant DNA technology. They are used in recombinant DNA technology. They are not used in recombinant DNA technology.

(2) Nomenclature of Restriction Enzymes:

(i) Type II restriction enzymes are named for the bacterium from which they have been isolated,

(ii) The first letter used for the enzyme is the first letter of the bacterium’s genus name (in italics),

(iii) Then comes the first two letters of its species (also in italics),

(iv) The fourth letter of the name of enzyme is first letter of the strain. It is written in capital,

(v) The end of the name indicates the order in which the enzyme was isolated. It is written in Roman number. For example, the enzyme Eco R1 was isolated from the bacterium Escherichia coli RY13. Enzyme Eco R1 is named as follows.

The capital letter E comes from the genus Escherichia. The letters со. are from the species coli. The letter R is from RY13 (strain). The Roman number 1 indicates that it was the first enzyme isolated from the bacterium E. coli RY13. The discovery of restriction endonuclease enzymes led to Nobel Prize for W. Arber, H. Smith and D. Nathan in 1978.

Some examples of type II enzymes are given in the table 11.1 showing names of restriction enzymes (endonucleases), source (organism from where they have been isolated), their recognition sequence and site of cleavage.

(3) Functioning of Restriction Endonuclease:

The foundations of recombinant DNA (rDNA) were laid by the discovery of restriction enzymes. These enzymes are present in many bacteria where they function as a part of their defence mechanism called the Restric­tion Modification System. Molecular basis of this system was explained first by Wemer Arber in 1965.

The Restriction-Modification system consists of two components

(i) A restriction enzyme which identifies the introduced foreign DNA and cuts into pieces called restriction endonucleases. The term ‘restriction’ refers to the function of these enzymes in restricting the propagation of foreign DNA of bacteriophages (viruses that attack bacteria) in the host bacterium,

(ii) The second component is a modification enzyme that adds a methyl group to one or two bases usually ‘within’ the sequence reorganized by the restriction enzyme.

Once a base in a DNA sequence is modified by addition of a methyl group, the restriction enzymes fail to recognize and could not cut that DNA. This is how a bacterium modifies and therefore, protects its own chromosomal DNA from cleavage by these restriction enzymes.

The first restriction endonuclease was Hin d II (hin-dee-two). Its functioning depended on a specific DNA nucleotide sequence. It was isolated from Haemophilus influenza Rd. It was found that Hin d II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs.

This specific base sequence is known as the recog­nition sequence for Hin d II. It produces blunt ends. Besides Hin d II, today we know more than 900 restriction enzymes that have been isolated from over 230 strains of bacteria each of which recognises different recognition sequences.

(4) Palindromic Nucleotide Sequence:

The restriction endonuclease inspects the length of a DNA sequence. Once it recognises specific sequence, it binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar phosphate back bones. Special sequence in the DNA recognised by restriction endonuclease is called palindromic nucleotide sequence.

Restriction endonuclease recognizes palindromic sequences in DNA and cuts them. The palindromes are groups of letters that form the same words when read in both directions forward and backward.

The palindromes in DNA are base pair sequences that are the same when read forward (left to right) or backward (right to left) from a central axis of symmetry. For example, the following sequences read the same on the two strands in 5’—> 3′ direction. This is also true when we read in the 3’—> 5′ direction.

Restriction enzymes cut the strand of DNA a little away from the centre of the palin­drome sites but between the same two bases of the opposite strands. This leaves single stranded unpaired bases at cut ends. These ends with unpaired bases are called sticky ends or cohesive ends (Fig 11.3). The latter are named so because they form hydrogen bonds with their complementary cut counter parts. The sticky ends facilitate the action of the enzyme DNA ligase.

Gel Electrophoresis (Separation and Iso­lation of DNA Fragments):

After the cutting of DNA by restriction enzymes, fragments of DNA are formed. Separation of DNA fragments according to their size or length is done by a technique called gel electrophoresis developed by A. Tiselius in 1937. Electrophoresis is a tech­nique of separation of molecules such as DNA, RNA or protein on the basis of their size, under the influence of an electrical field, so that they migrate in the direction of electrode bearing the opposite charge, viz., positively charged molecules move towards cathode (-ve electrode) and negatively charged mol­ecules travel towards anode (+ve elec­trode) through a medium/matrix.

Electrophoresis performed in a gel matrix, so that molecules of similar elec­tric charge can be separated on the basis of size, is called gel electrophoresis. Now a days the most commonly used matrix is agarose which is a polysac­charide extracted from sea weeds. DNA fragments separate according to size through the pores of agarose gel. Hence the smaller the fragment size the farther it moves. Agarose dissolves in hot water when this solution is cooled, double helices form and become arranged laterally and produce thick filaments. These filaments become cross- linked to form the gel (Fig. 11.5). Pore size depends on agarose concentration.

The separated DNA fragments can be seen only after staining the DNA with a compound known as ethidium bromide (EtBr) followed by exposure to UV radiation as bright orange coloured bands. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This process is called as elution (removal of adsorbent). These purified DNA fragments are used in constructing recombinant DNA by linking them with cloning vectors.

(B) Cloning Vectors (Vehicle DNA or carrier of DNA):

The vectors are DNA molecules that can carry a foreign DNA segment and replicate inside the host cell. Vectors may be plasmids, a bacteriophages (viruses that attack bacteria), cosmids, Yeast artificial chromosomes (YACs), Bacterial artificial chromosomes (BACs) and viruses. There are also some shuttle vectors. Out of these vectors, the most commonly used cloning vectors are plasmids and viruses.

Plasmids were discovered by Willium Hays and Joshua Lederberg (1952). These are extra-chromosomal, self-replicating, usually circular, double-stranded DNA molecules, found naturally in many bacteria and also in some yeast. Although plasmids are usually not essential for normal cell growth and division, they often confer some traits on the host organism, for example, resistance to certain antibiotics or toxins that can be a selective advantage under certain conditions.

The plasmid molecules may be present as 1 or 2 copies or in multiple copies (500-700) inside the host organ­ism. These naturally occurring plasmids have been modified to serve as vectors in the laboratory. The most widely used, versatile, easily manipu­lated vector pBR 322 is an ideal plasmid vector.

pBR322 Vector (Fig. 11.7):

This was the first artificial cloning vector constructed in 1977 by Boliver and Rodriguez. It is widely used in gene cloning experiments.

p — denotes that it is a plasmid

BR — stands for Boliver and Rodriguez who constructed this plasmid

322 — is a number given to distin­guish this plasmid from others developed in the same laboratory. For example, there are plasmids pBR 325, pBR 327, pBR 328, pBR 345 etc.

Features that is required to facilitate cloning into a Vector:

(i) Origin of Replication (Ori):

Origin of replication (Ori) is a specific sequence of DNA bases which is responsible for initiating replication. A prokaryotic DNA has a single origin of replication while eukaryotic DNA may have more than one origin of replication.

(ii) Selectable Markers:

Some genes called “selectable markers” help in selecting those host cells which contain the vectors (trans-formants) and eliminating the non­-trans formants. Transformation is a process through which a piece of DNA is introduced in a host bacterium. Generally, the genes encoding resistance to antibiotics such as tetracy­cline, ampicillin, kanamycin or chloramphenicol etc. are useful selectable markers for E. coli. The common E. coli cells are not resistant against any of these antibiotics. Plasmid pBR 322 has two resistance genes — ampicillin resistance (amp R ) and tetracyclin resistance (tet R ) which are considered useful for selectable markers.

(iii) Cloning Sites (Recognition Sites):

Plasmid pBR 322 has a variety of unique rec­ognition sites for restriction endonucleases. Two unique sites, Pst I and Pvu I are located within the amp R gene and Bam HI, Sal I, etc. are within tet R gene (Fig 11.7). Some other unique restriction sites are Eco RI, Cla I, Hind III, Pvu II, rop codes for the proteins involved in the replication of the plasmid.

The presence of restriction sites within the markers tet R and аmр R permits an easy selection for cells transformed with the recombinant pBR322. Insertion of the DNA frag­ment into the plasmid using enzyme Pst I or Pvu I places the DNA insert within the gene amp R this makes аmр R non-functional. Bacterial cells containing such a recombinant pBR322 will be unable to grow in the presence of ampicillin, but will grow on tetracycline. Similarly, when restriction enzyme Bam HI or Sal I is used, the DNA insert is placed within the gene tet r making it non-functional. Bacterial cells possessing such a recombinant pBR322 will, therefore, grow on ampicillin but not on tetracycline.

Due to inactivation of antibiotics, selection of recombinants becomes burdensome pro­cess because it requires simultaneous plating on two plates having different antibiotics. Thus, alternative selectable marker is developed to differentiate recombinants and non-recombi­nants on the basis of their ability to produce colour in the presence of a chromogenic substance. Now a recombinant DNA is inserted in the coding sequence of an enzyme β- galactosidase.

This casues inactivation of the enzyme which is called insertional inacti­vation. If the plasmid in the bacterium does not have an insert, the presence of a chromoge­nic substrate gives blue coloured colonies. Presence of insert results into insertional inacti­vation of the p-galactosidase and, therefore, the colonies do not produce any colour, these colonies are marked as recombinant colonies.

(iv) Vectors for Cloning Genes in Plants and Animals:

A soil-inhabiting plant bacte­rium, Agrobacteriun tumefaciens, a pathogen (disease causing agent) of several dicot plants is able to transfer a piece of DNA known as ‘T-DNA’. The T-DNA causes tumours. The tumours are called crown galls. Tumour formation is induced by Ti plasmid (Ti for tumour inducing).

As gene transfer occurs without human effort, the bacterium is called natural genetic engineer of plants. Similarly retroviruses (cause leukosis or sarcoma types of cancer) in animals including humans are able to change normal cells into cancerous cells.

The tumour including Ti plasmid of Agrobacterium tumefaciens have been modified into cloning vector which is not pathogenic to the plants, however, it is still able to use the procedure to deliver genes of our interest into various plants. Similarly retroviruses are used to carry desirable genes into animal cells. Thus once a gene or DNA fragment is joined to a suitable vector it is transferred into a bacterial plant or animal host where it undergoes multiplication.

(2) Bacteriophage Vectors:

Bacteriophages (are simply phages) are viruses that attack bacterial cells by injecting their DNA into these cells. The injected DNA is selectively rep­licated and expressed in the host bacterial cell resulting in a number of phages which burst out of the cell (lytic pathway) and reinfect neighbouring cells.

This ability to transfer DNA from the phage genome to specific bacterial hosts during the process of bacterial infection gave scientists the idea that specially designed phage vectors would be useful tools for gene cloning experiments. Several bacteriophages are being used as cloning vectors but most commonly used are lambda (X) phage and M13 phage.

(a) Lambda Phage Vector:

It has a double-stranded, linear DNA genome of 48, 502bp, in which the 12 bases at each end are unpaired but complementary. These ends are, therefore, sticky or cohesive and are referred to as the cos sites (cohesive end sites). These sites are important for packaging DNA into phage heads. The lambda genome remains linear in the phage head, but within E. coli cells the two cohesive ends join to form a circular molecule necessary for replication. These vectors allow cloning of dna fragments upto 23 Kb length (= Kilobase— is a unit designating the length of a nucleic acid sequence, e.g., 1 Kb = 1000 nucleotide long base sequence).

It is filamentous phage which infects E. coli having F-pili. Its genome is a single stranded, circular DNA of 6407 bp. Foreign DNA can be inserted into it without disrupting any of the essential genes. After the Ml 3 phage DNA enters the bacterial cell, it is converted to a double-stranded molecule known as the replicative form or RF, which replicates until there are 100 copies in the cell and single-stranded copies of the genome are produced and extruded from the cell as M13 particles.

The major advantages of developing vectors based on M13 are that its genome is less than 10Kb length. The RF can be purified and manipulated exactly like a plasmid. In addition, genes cloned in M13 based vectors can be obtained in the form of single-stranded DNA.

Why are bacteriophage vectors more advantageous than plasmid vectors?

(i) Bac­teriophage vectors can be used for large DNA fragments and

(ii) These can easily be detected at the time of cloning experiments.

(C) Competent Host (For Transformation with Recombinant DNA):

(a) DNA Mediated Gene Transfer (Vector Mediated Gene Transfer):

Competent host is essential for transformation with recombinant DNA (Fig. 11.12). Transformation “a process by which a cell takes up naked DNA fragment from the environment, incorporates it into its own chromosomal DNA and finally expresses the trait controlled by the incoming DNA”. The tools described earlier in this chapter will result in the generation of recombinant DNA (rDNA) molecules in the laboratory. Finally, the propagation of these DNA molecules must occur inside a living system or a host.

Many kinds of host cells, including E. coli, yeast, animal and plant cells, are available for genetic engineering and the kind of host cell to be used mainly depends on the aim of the cloning experiment. For the expression of some eukaryotic proteins, eukaryotic cells may be the preferred hosts. Yeasts have been used extensively for functional expression of eukaryotic genes because they offer several advan­tages. Yeasts are the simplest eukaryotic organisms and like bacteria are single-celled, genetically well-characterized, easy to grow and manipulate.

They can be grown readily in both small culture vessels and large scale bioreactors. Plant and animal 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. Since DNA is a hydrophilic molecule, it cannot pass through membranes, so the bacterial cells must be made capable to take up DNA.

This is done by treating them with a specific concentration of a divalent cation, such as Calcium which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA (rDNA) can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.

Transfer of DNA into eukaryotic cell is called transfection.

(b) Direct or Vector less Gene Transfer:

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

In this method foreign DNA is directly injected into the nucleus of animal cell or plant cell by using micro needles or micro pipettes. It is used in oocytes, eggs and embryo. Alec Jeffreys (1993) of Human Genome Centre, Michigan University U.S.A has cured mice that inherited a neuromuscu­lar disease which is like muscular dystrophy of humans.

Electroporation is the formation of temporary pores in the plasma membrane of host cells by using lysozyme or calcium chloride. These pores are used for introduction of foreign DNA.

3. Chemical Mediated Gene Transfer:

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

4. Biolistic Method or Gene gun Method:

Biolistic is a means of introducing DNA into cells that involves bombardment of cells with high-velocity micro projectiles coated with DNA. In biolistic method tungsten or gold particles, coated with foreign DNA are born barded into target cells at a very high velocity. Although this method is suitable for plants yet this technique is also used to insert genes into animal that promote tissue repair into cells (particularly cancer of mouth) near wounds.

This method failed to make an impression in treat­ment of genetic disorder but made great impact in the field of vaccine development.

After being introduced briefly to the tools in re­combinant DNA let us describe the processes to create recombinant DNA.