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Genetic engineering
Post: #1

Genetic programming has recently emerged as an important paradigm for automatic generation of computer programs. GP combines metaphors drawn from biological evolution with computer science techniques in order to produce algorithms and programs automatically.From the very beginning, man has tried to develop machines that can replace the need for human beings for many applications; machines that require very little support of human beings. Research is going on to develop machines that can produce high artificial components in the results, with least human intelligence. In this context, the GP assumes a special significance.
Over the past decade the artificial evolution of computer code has become a rapidly spreading technology with many ramifications. Originally conceived as a means to enforce computer intelligence, it has now spread into many areas of machine learning and is starting to conquer many areas.
Genetic programming has a recently emerged as an important paradigm for automatic generation of computer programs. GP combines metaphors drawn from biological evolution with computer science techniques in order to produce algorithms and programs automatically.
In the long run the Genetic Programming will revolutionalize program development. Present methods do not mature enough for deployment as automatic programming systems. Nevertheless, GP has already made inroads into automatic programming and will continue to do so
Post: #2
Presented By:Mayank Bhardwaj
Genetic Engineering

Historical Background
Father of Genetics – Gregor John Mendel
- Formulated set of rules to explain inheritance of biological characteristics.
- Basic assumption: Each heritable property is controlled by a factor, Gene.
- Rediscovery of Mendel’s law in 1900 marked birth of Genetics.
- Gene reside on chromosome: W. Sutton (1903)
- Experimental Backing: T.H. Morgan(1910)
- DNA as genetic material: Avery, McCarty, McLeod(1944), Hershey, Chase(1952).
- Between 1952-1966: Structure of DNA proposed, Genetic code cracked, Process of transcription and translation described.
Until decade of 1960 experimental techniques were not sophisticated enough to study gene.
- 1971 to 1973: Revolution in experimental biology.
# Recombinant DNA technology
# Genetic Engineering
Genetic Engineering A Biotechnological Revolution
The synonyms-
Recombinant DNA technology
R-DNA technology
Gene cloning
Molecular cloning

The Applications
Development of GMO and GEO.
Gene therapy (e.g. HGH, Human insulin).
Improving nutritional value of food.
Improving storage life of food.
Controlling water and air pollution.
DNA fingerprinting.

Gene cloning General process
Fragment of DNA with desired gene inserted to circular DNA molecule (vector) to produce chimaera / r-DNA
Vector act as vehicle for transferring gene to host cell ( gen. bacterium)
Within host cell, vector multiplies producing identical copies. Host cell itself divides and passes r-DNA to progeny.
Copies of desired genes are further extracted by selection and screening process.

Gene cloning Main Steps
Isolation and purification of DNA segments.

Addition of desired gene to vector.

Insertion of recombinant plasmid to host cell.

Selection and screening of clones of r-DNA

Gene cloning Basic Requirements
DNA purification from living cells for desired genes and vectors.
Vectors to carry the desired gene.
DNA manipulating enzymes.
Recombinant Gene transferring modes.
Recombinant Gene selection and screening modes.

Gene cloning 1. DNA purification
Two distinct kind of DNA are purified
a). Total cell DNA: i.e. desired DNA,
donor may be bacteria, animal, plant or any other cell.
Methods used are
- Centrifugation
- Phenol extraction
- Ethanol precipitation
- CTAB method for DNA from plant cell.

Gene cloning 1. DNA purification
B). Vector DNA : vehicle of desired DNA,
Cultures are prepared from plasmid or phages depending upon requirement.
methods used are
- Anion exchange column chromatography
- Alkaline denaturation method
- CsCl density gradient centrifugation
- PEG precipitation ( for phages).

Gene cloning 2. Vectors
It is a DNA molecule carrying foreign DNA to host cell, where it replicates producing its identical copies with foreign DNA.
- Also called Vehicle.
Types of Vector:
Phage/ Phasmid
Yeast artificial chromosome
Bacterial artificial chromosome

Gene cloning 2 a) Vector-Plasmid
Extra-chromosomal, circular DNA in bacteria, having self replicating abilities outside the host cell.
Cloning limit- 0.1 to 10KB.

Gene cloning 2 a) Vector-Plasmid-Types
Classification based on main characteristics coded by plasmid genes.
Fertility (F) plasmid: carry tra genes, have ability for conjugal transfer of plasmid. e.g. F of E.Coli
Resistance ® plasmid: carry gene for antibiotic resistance for host bacterium. e.g. RP4 of Pseudomonas.
Col Plasmid: Colicin code-protein killing other bacteria. E.g. ColE1 of E.Coli.
Degradatice plasmid : allow host bacterium to metabolize unusual molecule as toulene, salicylic acid.
e.g. TOL of Pseudomonas putida.

Gene cloning 2 b) Vector – Phage/ Phasmid
Virus infecting bacteria.
Affect bacteria by lytic and lysogenic cycles.
Although many varieties are there, mainly used are M13 and λ bacteriophage.

Gene cloning 2 b) Vector- Phage/ Phasmid
General pattern of infection
Phage particle attach outside of bacterium cell and injects its DNA.
Phage DNA molecule is replicated inside cell, coding is done by specific number of genes.
Rest genes directs synthesis of capsid, new phage particles are assembled and released from bacterium.

Gene cloning 2 c) Vectors- Cosmid
Circular, extra-chromosomal DNA molecules with combined features of plasmid and phage.
Cloning limit 35-50 times then phage and plasmid.

Gene cloning 2 d) Vectors-BAC
Bacterial artificial chromosome are small pieces of episomal DNA giving conjugation initiating ability to bacteria.
Have cloning limit of 75-300kb.

Gene cloning 2 d) Vectors- YAC
Yeast artificial chromosome replicates in yeast cells.
Have recognition sites for restriction enzymes.

Gene cloning 3. DNA manipulating enzymes
These enzymes are classified in 5 categories.
Nuclease: To cut, shorten, degrade nucleic acids.
a. Exonuclease- can remove one nucleotide at a time from DNA.
b. Endonuclease –Can break internal bonds in DNA molecules
Ligases : Join nucleic acid molecules.
Polymerase : Make copies of molecules.
Modifying enzymes: Remove or add chemical groups.
Topoisomerase : Remove or add supercoils from covalently closed circular DNA.

Gene cloning 3. DNA manipulating enzymes
Restriction Endonuclease
Known as DNA scissors.
These are highly specific enzymes that produce internal cuts in DNA.
They act specifically on recognition sites only

Gene cloning 3. DNA manipulating enzymes
Restriction Endonuclease- Types

Type-1 Restriction endonuclease e.g. Eco RI
not used in gene cloning.

Type-2 Restriction endonuclease e.g. Hind II, used in gene cloning.

Type-3 Restriction endonuclease e.g. Eco PI
not used in gene cloning.

Gene cloning 3. DNA manipulating enzymes
DNA ligase
Function is to join vector DNA and desired DNA
Purified from t4 phage infected E.Coli bacteria.
Can join blunt ends and sticky ends with later at higher frequency.

Gene cloning 4. Rec. gene transferring modes
Recombinant gene is transferred to host cell by following methods :



Gene cloning 4. Rec. gene transferring mode
Usually bacterial cell can uptake DNA easily during transformation, but not all species are efficient.
- Electroporation : Giving electric shock of 100-200v and making holes in cell.
- CaCl2 : In presence of Ca 2+ cell wall become permeable to plasmid DNA, heat treatment of 420C for 30-120 sec causes DNA to enter cell.
_ Lipofection : vesicles with desired plasmid is allowed to fuse with cell membrane leading to insertion of gene.

Gene cloning 5. Rec. gene selection and screening mode

Selection : Allowing desired cell to replicate while preventing un- desired cell from replicating.

Screening : Checking clones grown after selection for desired and required properties.

Gene cloning 5. Rec. gene selection and screening mode
Selection and screening techniques:
Antibiotic sensitivity
Enzyme activity
Southern blotting
Colony hybridization

Gene cloning 5. Rec. gene selection and screening mode
Antibiotic sensitivity
If a antibiotic resistance gene is transferred as desired trait, then bacterial colony will grow even if they are treated with antibiotic.

Gene cloning 5. Rec. gene selection and screening mode
Enzyme activity:
Normal cells are able to synthesize β-gal.
Recombinants cannot synthesize by inactivation of β-gal synthesizing gene.
Screening done by X- gal and IPTG.
Recombinant show white color, non-rec shows blue color.

Gene cloning 5. Rec. gene selection and screening mode
Colony Hybridization
Bacteria is transferred from culture plate to nitro-cellulose filter.
Bacteria lysed and DNA denatured.
Incubate and hybridize desired gene with radioactively labeled nucleic acid probe.
Autoradiography is done to expose colony with hybridized probe.
Post: #3
[attachment=14144] What is Genetic Engineering?
The genes present in the body of all living organisms helps determine the organism’s habits. Genetic engineering is defined as a set of technologies that are used to change the genetic makeup of cells and move the genes from one species to another to produce new organisms. The techniques used are highly sophisticated manipulations of genetic material and other biologically important chemicals.
Genetic engineering is the the use of various methods to manipulate the deoxyribonucleic acid (DNA)of cells to produce biological products or to change hereditary traits. Techniques used include using needles to insert DNA into an ovum, hybridomas (hybrids of cancer cells and of cells that make a desired antibody), and recombinant DNA, in which the DNA of a desired gene is inserted into the DNA of a bacterium. The bacterium then reproduces itself, yielding more of the desired gene. Another type is the polymerase chain reaction (PCR) which referrs to a lab process in which a particular DNA segment is quickly replicated to create a large, easily analyzable sample. The process makes perfect copies of DNA fragments and is used in DNA fingerprinting.
Genetic engineering is the alteration of genetic code by artificial means, and is therefore different from traditional selective breeding.
Genetic engineering examples include taking the gene that programs poison in the tail of a scorpion, and combining it with a cabbage. These genetically modified cabbages kill caterpillers because they have learned to grow scorpion poison (insecticide) in their sap.
Genetic engineering also includes insertion of human genes into sheep so that they secrete alpha-1 antitrypsin in their milk - a useful substance in treating some cases of lung disease.
Genetic engineering has created a chicken with four legs and no wings.
Genetic engineering has created a goat with spider genes that creates "silk" in its milk.
Genetic engineering works because there is one language of life: human genes work in bacteria, monkey genes work in mice and earthworms. Tree genes work in bananas and frog genes work in rice. There is no limit in theory to the potential ofgenetic engineering.
Genetic engineering has given us the power to alter the very basis of life on earth.
Genetic engineering has been said to be no different than ancient breeding methods but this is untrue. For a start, breeding or cross-breeding, or in-breeding (for example to make pedigree dogs) all work by using the same species. In contrast genetic engineering allows us to combine fish, mouse, human and insect genes in the same person or animal.
Genetic engineering therefore has few limits - except our imagination, and our moral orethical code.
Genetic engineering makes the whole digital revolution look nothing. Digital technology changes what we do. Genetic engineering has the power to change who we are.
Human cloning is a type of genetic engineering, but is not the same as truegenetic manipulation. In human cloning, the aim is to duplicate the genes of an existing person so that an identical set is inside a human egg. The result is intended to be a cloned twin, perhaps of a dead child. Genetic engineering in its fullest form would result in the child produced having unique genes - as a result of laboratory interference, and therefore the child will not be an identikit twin.
Genetic engineering could create crops that grow in desert heat, or without fertiliser. Genetic engineering could make bananas or other fruit which contain vaccines or othermedical products.
Genetic engineering will alter the basis of life on earth - permanently - unless controlled. This could happen if - say - mutant viruses, or bacteria, or fish or reptiles are released into the general environment.
If genetic engineering is defined as changing an organism's DNA to make it more beneficial, genetic engineering has been going on for a very, very long time in the form of selective breeding. However, actually going into a cell and changing its genome by inserting or removing DNA is a very new technology.
Ancient History
Selective breeding has been going on for countless generations. In fact, it is even mentioned in the Bible (Genesis 30:25 - 43). In the account, Jacob was employed as a shepherd under his father-in-law Laban. Instead of receiving wages, Jacob received the black, streaked, and spotted sheep, and Laban kept all the white sheep. Jacob craftily arranged for his black sheep to mate with Laban's white sheep, producing streaked and spotted sheep. Jacob did so well with this scheme that Laban's family began to get mad at Jacob, and he eventually had to leave.
Selective breeding is effective enough if the goal is to maintain or gradually improve a group of animals. Over the decades, selective breeding has brought us improved strains of cattle and specialized breeds of dogs. However, these advances have taken hundreds of years to effect. In addition to the time concerns, it is often impossible to know which traits will be transferred to the offspring.
Selective breeding is a long, tedious process that has its limits. It is impossible through selective breeding to mix traits from two totally different species. If a junkyard owner wanted a guard dog that could squirt ink like an octopus, he would be unable to create such an animal. It is physically impossible, because the genetics of life are such that traits from two different organisms cannot be mixed. That is where genetic engineering comes in.
The Progress
Modern genetic engineering began in 1973 when Herbert Boyer and Stanley Cohen used enzymes to cut a bacteria plasmid and insert another strand of DNA in the gap. Both bits of DNA were from the same type of bacteria, but this milestone, the invention of recombinant DNA technology, offered a window into the previously impossible -- the mixing of traits between totally dissimilar organisms. To prove that this was possible, Cohen and Boyer used the same process to put a bit of frog DNA into a bacteria.
Since 1973, this technology has been made more controllable by the discovery of new enzymes to cut the DNA differently and by mapping the genetic code of different organisms. Now that we have a better idea of what part of the genetic code does what, we have been able to make bacteria that produce human insulin for diabetics (previously came from livestock), as well as EPO for people on kidney dialysis (previously came from urine of people in third world countries with ringworm).
In 1990, a young child with an extremely poor immune system recieved genetic therapy. Some of her white blood cells were genetically manipulated and re-introduced into her bloodstream while she watched Sesame Street. These new cells have taken over for the original, weak white cells, and her immune system now works properly. Although relatively few people have had their cells genetically altered, these advances have made the prospect of mainstream genetic medicine seem more likely.
The Promise
Genetic engineers hope that with enough knowledge and experimentation, it will be possible in the future to create "made-to-order" organisms. This will lead to new innovations, possibly including custom bacteria to clean up chemical spills, or fruit trees that bear different kinds of fruit in different seasons. Any trait occurring in nature can theoretically be mixed with any other to form a totally new organism that would not otherwise occur in nature.
[b]Current Status [/b]
As of late summer of 1998, scientists are able to add simple traits to organisms. They cannot create custom-made animals. They cannot always predict how traits will interact. Before phenomenally new advances can be made, scientists have to learn how to affect cells' DNA with pin-point accuracy, without affecting other traits. Advances like genetic correction for nearsightedness are a long way off. The power of science is limited to knowledge about genetics, gene locations, and trait interactions, but as knowledge grows, so will scientists' abilities to manipulate life.

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