Biotechnoloy Introduction and application

Table of Content

List of Figures……..……………………………………………3 Intordoction……………………………………………………..4 1. Host for cloning vector………………………………………. 5 1.1 Prokaryotic hosts ……………………………………………5 1.2 Eukaryotic hosts ……………………………………………6 2. Transfection of Eukaryotic cells ……………………………..8 2.1 electroporation ……………………………………………...8 2.2 particle gun ………………………………………………….9 3. Finding the right clone ……………………………………….10 3.1 Antibody as a method of detecting the protein …………….10

3.2 Nucleic acid probe …………………………………………..11 3.2.1 Preparing the filters ………………………………………..11 3.2.2 Probing the filters ………………………………………….12 4. Specialized vectors ……………………………………...…….13 4.1 Shuttle vector ..………………………………………………13 4.2 Expression vector ………………………………………..…..13 4.3 Regulation of transcription from expression vector ……...….13 5. Reporter genes ………………………………………………..14 6. Expression of mammalian genes in bacteria …………….…...15 7. Protein folding and stability …………………………...……..17 8. Examples of biotechnological applications …………...……...17 8.1 Making proteins ………………………………………….….17 8.2 Production of enzymes ………………………………………17 8.3 The BST recombinant bovine somatotropin ……………...…18 8.4 Therapeutics …………………………………………………18 8.4.1 Gene therapy …………………………………….………...18 8.4.2 prouduct insulin …………………………………………...18 8.4.3 Cell Transplants ………………………...…………………20

8.4.4 Biotechnology Vaccine Production ……….………………20 9. Agricultural Production Applications ……………..…………21 Definitions……………………………………..………………...23 References …………………………………….……………..… 24

List of Figures

Figure (1): cloning host…………………………………………...7 Figure (2): cells under elctroporation …………………………….8 Figure (3): elctroporation machine ……………………………….9 Figure (4): shotgun………………………………………………..9 Figure (5): Antibody as a method of detecting the protein………11

Figure (6): screening a DNA library by hybridization with a DNA……………………………………………………………...13 Figure (7): the isolated mRNA is used to make complementary DNA (cDNA) by means of reverse transcription………………..16 Figure (8): product insulin by biotechnology……………………19 Figure (9): biotechnology application……………………………22

Intordoction
Biotechnology is the use of living organisms to carry out chemical processes for industrial or commercial application .These techniques make it possible to isolate and manipulation DNA as well as to control its expression . The basic techniques of genetic engineering like the techniques of modern microbial genetics . Much of genetic engineering is based on molecular cloning . in molecular cloning a double-stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host ,Commonly employed cloning vector include plasmids and bacteriophags .

The techniques of genetic engineering are based on fundamental discoveries in molecular genetics and biochemistry. Successful genetic engineering depends not only on being able to carry out molecular cloning, but also knowledge of replication, transcription, and translation.

1.Host for cloning vector
To obtain a large amounts of cloned DNA, the host should be grow rapidly in an expensive culture medium , non pathogenic , be capable of taking up DNA be genetically stable in culture and have the appropriate enzymes to allow replication of the vector . The most useful host for cloning are typically microorganisms that grow well and for which we have much information . these include the bacteria E. coli , B.subtilis and the yeast sccharomyces cerevisiae , a compleat genome sequences for these organisms are a viable .

1.1 Prokaryotic hosts
The bacterium E. coli fulfils these requirements and is used in many cloning protocols , E. coli has been studied in great detail and many different strains were isolated by microbial geneticists as they investigated the genetic

mechanisms of this prokaryotic organism. Such studies provided the essential background information on which genetic engineering is based. E. coli is a gram-negative bacterium with a single chromosome packed into a compact structure known as the nucleoid. The genome size is some 4.6 × 106 base pairs, and the complete sequence is now known. The processes of gene expression (transcription and translation) are coupled, with the newly synthesized mRNA being immediately available for translation. There is no post-transcriptional modification of the primary transcript as is commonly found in eukaryotic cells. E. coli can therefore be considered as one of the simplest host cells .Much of the gene cloning that is carried out routinely in laboratories involves the use of E. coli hosts, with many genetically different strains available for specific applications. In addition to E. coli, other bacteria may be used as hosts for gene cloning experiments, with examples including species of B.subtilis is not potential pathogen ,dose not produce endotoxin dose produce spores . several plasmid and phage suitable for cloning have been developed and transformation is a well developed procedure in B.subtilis disadvantage of using B.subtilis include plasmid instability . it can be difficult to maintain plasmid replication over many subcltures of the organism also, Foreign

DNA is not well maintained in B.subtilis cells and so the cloned DNA is often unexpectedly lost .

1.2 Eukaryotic hosts
One disadvantage of using an organism such as E. coli as a host for cloning is that it is a prokaryote, and therefore lacks the membrane bound nucleus (and other organelles) found in eukaryotic cells. This means that certain eukaryotic genes may not function in E. coli as they would in their normal environment, which can hamper their isolation by selection mechanisms that depend on gene expression .Prokaryotic host cells have certain limitations when the cloning and expression of genes from eukaryotes is the aim of the procedure . Also, if the production of a eukaryotic protein is the desired outcome of a cloning experiment, it may not be easy to ensure that a prokaryotic host produces a fully functional protein. Eukaryotic cells range from microbes, such as yeast and algae, to cells from complex multicellular organisms, such as ourselves. The microbial cells have many of the characteristics of bacteria with regard to ease of growth and availability of mutants. Higher eukaryotes present a different set of problems to the genetic engineer, many of which require specialized solutions. Often the aim of a gene manipulation experiment in a higher plant or animal is to alter the genetic makeup of the organism by creating a

transgenic, rather than to isolate a gene for further analysis or to produce large amounts of a particular protein. The yeast S .cerevisiae is one of the most commonly used eukaryotic microbes in genetic engineering. It has been used for centuries in the production of bread and beer and has been studied extensively. The organism is amenable to classical genetic analysis, and a range of mutant cell types is available. The yeast S. cerevisiae has about 3.5 times more DNA than E. coli. The complete genome sequence is now known. Other fungi that may be used for gene cloning experiments include A. nidulans and N. crassa. Plant and animal cells may also be used as hosts for gene manipulation experiments. Unicellular algae such as C. reinhardtii . All the advantages of microorganisms plus the Microbes (such as yeast) and functional organisation of plant cells, and their use in mammalian cell lines are two examples of eukaryotic host cells that have become widely used in gene manipulation. genetic manipulation will increase as they become widely Other (and cells are more studied. plant animal) usually

grown as cell cultures, which are much easier to manipulate than cells in a whole organism.

Figure (1): cloning host

2.Transfection of Eukaryotic cells
Eukaryotic microorganisms , animal and plant cells can take up DNA in a process that resembles bacterial transformation because the word transformation in mammalian cells is used to describe the conversion of cells to the malignant state . the introduction of DNA into mammalian cells has been called Transfection. Which is an important organism for genetic engineering Transfection at low efficiencies can be mediated by various methods .Because animal cells

do not have cell walls . in yeast transfection at low efficiencies can be mediated by various method like electroporation and particle gun.

2.1 electroporation
Widely applied to all types of eukaryotic cells and can beused whether or not the cell wall of the organism is removed . electroporation involves exposing host cells to pulsed electrical field in the presence of cloned DNA . the electrical treatment opens small pores in there membranes through which the DNA can enter . the pores generated are insufficient to cause cell damage or lysis but sufficient to allow cloned DNA to enter .

Figure (2) : cells under elctroporation

Figure (3) : elctroporation machine 2.2 particle gun
Operates some what like a sutgun a small steel cylinder containing a gunpowder charge is used to fire nuclic acid-coated particles at the cell, the particles bombard the cell piercing cell walls and membranes without actually killing the cell .

Figure (4): shotgun

3.Finding the right clone
Genetic engineering begins with the isolation of clone containing a gene of interest . Methods to clone DNA include making gene library from total genomic DNA or cloning a DNA fragment by PCR . one usually clones from PCR product when the gene of interest has already been identified and the goal is simply to obtain a single clone . One can select for host containing a plasmid vector by selecting for a vector marker such as antibiotic resistance , so that only these cells form colonies . colonies can also be screened for vectors that containing forging DNA inserts by looking for the inactivation of vector gene by different way .

3.1 Antibody as a method of detecting the protein

Antibodies can be used as detection reagents for the protein of interest . the protein product encoded by the cloned gene is the antigen , and this protein is used to produce an antibody in an experimental animal . since the antibody combine specifically with the antigen when the antigen is present in one or more colonies can be determined by observation the binding of the antibody .This method of detection and so thousands of clones may need to be examined this procedure using radio active detection system .

Figure (5): Antibody as a method of detecting the protein

3.2Nucleic acid probe
One of the other advantages of this type of labeling is that the methods of detecting the labeled probe often involve an amplification process so that you can detect the presence of small quantities of bound probe.

3.2.1Preparing the filters
Once you have labeled your DNA probe, you need to transfer the DNA from individual library members onto a solid support, in such a way that they can be related back to the original library member, we need to immobilize the proteins, produced before screening can begin This is done using a method analogous to the plaque lift (by each of the phage in our library, on a solid support, in such a way that we know which original plaque they were produced by. This technique ,often called a “plaque lift”) . the library is plated out and a small amount of bacteria from each colony is transferred to a sterile filter The filters are then taken through the series of treatments outlined in to produce filters with DNA from the bacterial colonies bound to them. Firstly, the bacteria are lysis by treatment with the detergent SDS and sodium hydroxide, to release the DNA, which will include chromosomal DNA as well as library DNA encoded on the plasmid.

The alkaline conditions will also denature the DNA making it singlestranded and available to hybridize with the probe molecules. Next the alkali has to be neutralized, otherwise it will interfere with annealing of the probe. DNA is covalently cross-linked to the filters before hybridization. It is bound to the filter by its sugar–phosphate backbone leaving the bases free to hybridize with the complementary bases of the DNA probe. Both nylon and nitrocellulose filters can be used in this procedure as DNA can be bound to both substrates. However, nylon filters are usually preferred as they are more robust than nitrocellulose and the DNA can be fixed to them either by heating or exposure to UV light. The same procedure can be used to screen a phage library, in which case the lysis step is not necessary as the phage plaques represent lysed bacteria from which the DNA has already been released.

3.2.2 Probing the filters
In DNA hybridization techniques it is important to use conditions where the probe will only bind to the specific DNA sequence it is designed to probe. In the case of colony hybridization this usually means using conditions of low stringency initially, in these conditions the probe will bind to many sequences even where there is only a partial match. Higher specificity is then achieved by a series of washes of increased stringency that

remove probe that is bound non-specifically. The usual ways of increasing stringency are to raise the temperature and either reduce the sodium chloride concentration, or increase the SDS concentration in the buffer. With oligonucleotide probes you can use higher stringency conditions with probes with higher melting temperatures as they bind more stably to their complementary sequences. If you are using degenerate oligonucleotides or probes derived from a homologous sequence you would decrease the stringency of the conditions compared with those you would use for a probe that was an exact complement to the sequence you are screening for.

Figure (6): screening a DNA library by hybridization with a DNA

4.Specialized vectors 4.1 Shuttle vectore

Allow cloaned DNA between unrelated organisms those a shuttle vector is a cloning vector that can be stably replicate in tow different organism .The importance in shuttle vector is that DNA cloned in one organisms can be replicated in a second host with out modifying the vector in any way. It is facilitated the expression of cloned DNA.

4.2 Expression vector
Vector that can be used not only to clone the desired gene but can also contain the necessary regulatory sequences so that expression of gene may be manipulated .

4.3Regulation of transcription from expression vector
Key element in an expression vector is a mechanism for transcriptional control . for high levels of expression it is essential to produce high levels of mRNA the expression vector must contain strong promoter that will function efficiently in the host and one that is correctly positioned so that it can permit the transcription in high level of the cloned gene . The expression vector is designed such that the cloned gene is fused to a promoter and operator region . this permits proper arrangement of the sequence of genetic elements : promoter – operator – ribosome binding site – structural gene , such that efficient transcription and translation occur . in most cases the operator and promoter cross pond to each other .

5.Reporter genes
Incorporated into vector because they encode proteins that are readily detected . these genes can be used to signal the presence or absent of a particular genetic element or its location . they can be fused to other genes or to the promoter of other genes or to the promoter of other genes or to the promoter of other genes so that expression can be studied . one can then detect colonies containing this reporter system on agar plates by their luminescence against a large background of other colonies

6.Expression of mammalian genes in bacteria
One approach to isolating a functional eukaryotic gene to clone it through its mature mRNA is not has any introns originally present have been removed. The isolated mRNA is used to make complementary DNA (cDNA) by means of reverse transcription .Once mRNA has been isolated it is necessary to convert the information to DNA . this is accomplished by use of the enzyme reverse transcriptase . this enzyme an essential component of retrovirus replication ,copies information from RNA into DNA a process called reverse transcription , reverse transcriptase require a primer in order for it to begin working . in cloning from messenger RNA an oligo-dt primer

is used that id complementary to the poly-A tail of the mRNA . the oligo-dt primer is hybridized with the mRNA and to this mixture , reveres transcriptase is added . The newly synthesized complementary DNA (cDNA) has hairpin loop at its end that is synthesized because after the enzyme completes copying the mRNA it starts to copy the newly synthesized DNA this hairpin loop provides a convenient primer for synthesis of the complementary strand of DNA . the resultant double-stranded DNA , with the hairpin loop intact , is then cleaved by a single strand-specific nuclease to produce the desired double strand DNA , one strand of which is complementary to the mRNA . this double-stranded DNA encode the gene of interest and can be. Inserted into a plasmid or other vector for cloning . The cDNA obtained in this way encodes the protein of interest and contains no introns . although there is a start codon , there are no promoters because these are not transcribed and therefore their sequence will not be In the mRNA .

Figure (7): the isolated mRNA is used to make complementary DNA (cDNA) by means of reverse transcription

7.Protein folding and stability

Some times when foreign proteins are massively over produced they form inclusion bodies are relatively easy to purify because of there size .the protein found in these bodies can be very difficult to solubilize . in many cases these bodies form because the proteins is incorrectly folded . one potential solution to this problem is to use a host that over produces molecular chaperons that aid to folded protein. folding problems can often besolved if the protein from the cloned gene is often besolved if the protein from the cloned gene is made as a fusion protein along with a protein encoded by the vector . Several special fusion vector are now available to encode fusion proteins .the cloned protein is then released from the fusion protein after purification by special proteases .

8.Examples of biotechnological applications 8.1 Making proteins
The synthesis and purification of proteins from cloned genes is one of the most important aspects of genetic manipulation, particularly where valuable therapeutic proteins are concerned. Many such proteins have already been

produced by recombinant DNA (rDNA) techniques and are already in widespread use .

8.2 Production of enzymE
In many cases the enzymee are prepared from natural sources, but in recent years there has been a move towards the use of enzymes produced by rDNA methods, where this is possible. In addition to the scientific problems of producing a recombinant-derived enzyme, there are economic factors to take into account, and in many cases the cost-benefit analysis The preparation of enzymes is a central part of biotechnology and ranges from the production of large amounts of low-cost makes the use of a recombinant enzyme unattractive. enzyme is the production of cheese. In cheese manufacture, rennet (also known as rennin, chymase, or chymosin) has been used as part of the process. Chymosin is a protease that is involved in the coagulation of milk casein following fermentation by lactic acid bacteria. It was traditionally prepared from animal (bovine or pig) or fungal sources recombinant-derived proteins in consumer products is the use of enzymes in washing powder. Proteases and lipases are commonly used to assist cleaning by degradation of protein and lipid-based staining. A recombinant lipase was developed in1988 by Novo Nordisk A/V (now known as Novozymes).

8.3 The BST recombinant bovine somatotropin
The BST gene was in fact one of the first mammalian genes to be cloned and expressed, using bacterial cells for production of the protein. Thus, the production of rBST at a commercial level, involving the basic science and technology transfer stages, was achieved without too much difficulty.

8.4 Therapeutics
Biotechnology will make possible improved versions of today’s therapeutic regimes as well as innovative treatments that would not be possible without these new techniques . 8.4.1Gene therapy presents an opportunity using DNA, or related molecules such as RNA, to treat diseases. For example, rather than giving daily injections of missing proteins, physicians could supply the patient’s body with an accurate instruction manual . a non defective gene correcting the genetic defect so the body itself makes the proteins. Other genetic diseases could be treated by using small pieces of RNA to block mutated genes. Only certain genetic diseases are enable to correction via replacement gene therapy. These are diseases caused by the lack of a protein, such as hemophilia and severe

combined immunodeficiency disease (SCID), commonly known as the “bubble boy disease.” Some children with SCID are being treated with gene therapy and enjoying relatively normal lives.

8.4.2 insulin product
many hormone are small protein these molecules are critical for the proper control of mammalian metabolism and have important therapeutic uses .insulin is a protein produced in the pancreas that is vital for regulation of glucose metabolism in the body . juvenile diabetes a disease characterized by insulin deficiency afficts millions of people . The obtine effective expression the synthesized insuln gene were inserted downstream from a sutible E.coli was synthesized as part of a fusion protein .an important advantage of making the fusion product protein is that the fusion was much more stable in E.coli than insulin it self .finally a codon for methonine was added at the junction joining the insulin gene to the upstream part of fusion gene . many tricks learned in the genetic engineering of insulin were applied to other product. insuline producting today stand as one of the crowing achievement of this technology . The first human protein made commercially using engineered bacteria was human insulin, but numerous other hormones and other human proteins are now being produced. Many proteins found in humans that were formerly

extremely expensive to produce because they were found in human tissues in only small amounts can now be made in very large amounts from the cloned gene in a suitable expression system. In addition to useful pharmaceuticals such as anticancer agents and immune modulators, even vaccines can be produced using genetic engineering.

Figu re (8): product insulin by biotechnology

8.4.3 Cell Transplants
scientists are investigating ways to use cell culture to increase the number of patients who might benefit from one organ donor. Liver cells grown in culture and implanted into patients kept them alive until1 diabetes,

researchers implanted insulin-producing cells from organ donors into the subjects’ livers. Eighty percent of the patients required no insulin injections one year after receiving pancreatic cells; after two years, 71 percent had no need for insulin injections. In another study, skeletal muscle cells from the subject repaired damage to cardiac muscle caused by a heart attack. Drugs for suppressing the immune response must be given if the transplanted cells are from someone other than the patient. Researchers are devising new ways to keep the immune system from attacking the transplanted cells. One method being used is cell encapsulation, which allows cells to secrete hormones or provide a specific metabolic function without being recognized by the immune system. As such, they can be implanted without rejection. Other researchers are genetically engineering cells to express a naturally occurring protein that selectively disables immune system cells that bind to it. Other conditions that could potentially be treated with cell transplants are cirrhosis, epilepsy and Parkinson’s Disease.

8.4.4 Biotechnology Vaccine Production
Most of the new vaccines consist only of the antigen ,not the actual microbe. The vaccine is made by inserting the gene that produces the antigen into a manufacturing cell, such as yeast. During the manufacturing process, which is similar to brewing beer, each yeast cell makes a perfect copy of

itself and the antigen gene. The antigen is later purified from the yeast cell culture. By isolating antigens and producing them in the laboratory, it is possible to make vaccines that cannot transmit the virus or bacterium itself. This method also increases the amount of vaccine that can be manufactured because biotechnology vaccines can be made without using live animals. Using these techniques of biotechnology, scientists have developed antigen-only vaccines against life-threatening diseases such as hepatitis B and meningitis. Recently researchers have discovered that injecting small pieces of DNA from microbes is sufficient for triggering antibody production. Such DNA vaccines could provide immunization against microbes for which we currently have no vaccines. Biotechnology is also broadening the vaccine concept beyond protection against infectious organisms. Various researchers are developing vaccines against diseases such as diabetes, chronic inflammatory disease, Alzheimer’s Disease and cancer. Various researchers are developing vaccines against diseases such as diabetes, chronic inflammatory disease, Alzheimer’s Disease and cancer.

9. Agricultural Production Applications
Many species of microorganisms interact with plants either as symbionts or as pathogens, and scientists have taken advantage of this intimate

relationship in their efforts to improve agricultural production through biotechnology. For example, plant-pathogenic bacteria, inactivated through the deliberate deletion of genes essential for pathogenesis (such as the ice nucleation gene) have been used successfully as competitors against natural pathogens. Similar efforts are being made to improve the properties – for example, the host range – of beneficial symbionts such as nitrogen-fixing bacteria. In most cases, however, the focus of improvement has been the genetic constitution of a given plant – that is, the production of transgenic plants. However, even these cases have exploited the natural capacity of the plant pathogen A. tumefaciens to introduce a portion of its plasmid DNA, TDNA, into plant cells.Theintroduction of exogenous genes fromother plants or microorganisms in this manner has resulted in the production of plants that are resistant to herbicides, insect pests, or viruses and that are now planted on a vast scale, totaling more than 80 million hectares worldwide. In the laboratory, some success also has been obtained in the creation of transgenic plants that are broadly pathogen resistant or salt tolerant. The next generation of transgenic plants will place more emphasis on the improvement of the quality of an agricultural product, and rice plants producing grains of higher nutritional values have already been obtained in the laboratory.

Fruits and vegetables with an improved shelf life and flowers with new and unexpected colors have also been successfully produced. Even the production of crops at higher yields may not be out of reach. A better understanding of the regulation of plant genes, however, is essential before these goals can be achieve .

Figure (9) biotechnology application

definitions
Biotechnology Number of technologies which use biological organisms to produce useful products, processes and services. Genetic engineering process where genetic material (DNA) is taken from one organism and inserted into the cells of another organism . Cloning Vector intentionally designed artificial DNA construct used by molecular biologists to amplify selected pieces of DNA inserted into the construct . Plasmid A small circular DNA molecule found in bacteria that replicates independently of the chromosome. Plasmids are used as cloning vectors.

REFRENCE
1- Nicholl.D, (2008) . An Introduction to Genetic Engineering.third edition. Cambridge University Press, New York 2- Walsh.G, (2004) BIOCHEMISTRY AND BIOTECHNOLOGY. Second Edition. John Wiley & Sons Ltd, England

3-NAJAFPOUR.G,(2007).BIOCHEMICALENGINEERING ANDBIOTECHNOLOGY. Elsevier 4- Walker.J and Rapley.R , (2002) . Molecular Biology and Bio technology. Fourth Edition. Royal Society of Chemistry 5- Lodge.J , Lund.P & Minchin.S,( 2007) . Gene Cloning Principles and Applications. Taylor & Francis, New York 6- Madigan.M and Martinko. J (2006). brock biology of microorganisms. Eleven editions. Pearson Prentice Hall, Inc 7- Kayser.O and Muller.R , (2004). Pharmaceutical Biotechnology Drug Discovery and Clinical Applications Pharmaceutical. Wiley-VCH Verlag GmbH & Co. KGaA

8- Brown.T , (2006). Gene cloning and DNA analysis: an introduction .fifth edition . Blackwell Publishing, Oxford

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