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 more

widely studied. Other plant (and animal) cells are 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 single-

stranded 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.

Figure (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.

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