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Genetic engineering

Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes. Genetic engineering is different from traditional breeding, where the organism's genes are manipulated indirectly. Genetic engineering uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Genetic engineering techniques have found some successes in numerous applications. Some examples are in improving crop technology, the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster ovary cells, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research.

The term "genetic engineering" was coined in Jack Williamson's science fiction novel Dragon's Island, published in 1951, two years before James Watson and Francis Crick showed that DNA could be the medium of transmission of genetic information.

Engineering

There are a number of ways through which genetic engineering is accomplished. Essentially, the process has five main steps:

1. Isolation of the genes of interest
2. Insertion of the genes into a transfer vector
3. Transfer of the vector to the organism to be modified
4. Transformation of the cells of the organism
5. Selection of the genetically modified organism (GMO) from those that have not been successfully modified.

Isolation is achieved by identifying the gene of interest that the scientist wishes to insert into the organism, usually using existing knowledge of the various functions of genes. DNA information can be obtained from cDNA or gDNA libraries, and amplified using PCR techniques. If necessary, i.e. for insertion of eukaryotic genomic DNA into prokaryotes, further modification may be carried out such as removal of introns or ligating prokaryotic promoters.

Insertion of a gene into a vector such as a plasmid can be done once the gene of interest is isolated. Other vectors can also be used, such as viral vectors, bacterial conjugation, liposomes, or even direct insertion using a gene gun. Restriction enzymes and ligases are of great use in this crucial step if it is being inserted into prokaryotic or viral vectors. Daniel Nathans and Hamilton Smith received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases.

Once the vector is obtained, it can be used to transform the target organism. Depending on the vector used, it can be complex or simple. For example, using raw DNA with gene guns is a fairly straightforward process but with low success rates, where the DNA is coated with molecules such as gold and fired directly into a cell. Other more complex methods, such as bacterial transformation or using viruses as vectors have higher success rates.

After transformation, the GMO can be selected from those that have failed to take up the vector in various ways. One method is screening with DNA probes that can stick to the gene of interest that was supposed to have been transplanted. Another is to package genes conferring resistance to certain chemicals such as antibiotics or herbicides into the vector. This chemical is then applied ensuring that only those cells that have taken up the vector will survive.

Applications

The first genetically engineered medicine was synthetic human insulin, approved by the United States Food and Drug Administration in 1982. Another early application of genetic engineering was to create human growth hormone as replacement for a compound that was previously extracted from human cadavers. In 1987 the FDA approved the first genetically engineered vaccine for humans, for hepatitis B. Since these early uses of the technology in medicine, the use of GM has gradually expanded to supply a number of other drugs and vaccines.

One of the best-known applications of genetic engineering is the creation of GMOs for food use (genetically modified foods); such foods resist insect pests, bacterial or fungal infection, resist herbicides to improve yield, have longer freshness than otherwise, or have superior nutritional value.

BACTERIA!

Agrobacterium Mechanism

Microinjection

Genetic Drift Simulation

What is Genetic Drift?

To begin with, let's examine a simple model of a population of fictional organisms called driftworms. In the following examples, the driftworms have only one gene, which controls skin color. Worms reproduce asexually and are connected to their parents by lines. In the population of five worms below, each worm gives rise to exactly one worm in the next generation. There are five alleles (skin colors) at generation 0 and the same five alleles at generation 4.

Note that the model above starts with a diverse population (5 worms, 5 alleles). What would the model look like if there were no diversity to begin with?

With no diversity in generation 0 and no forces of evolution acting on the population, the model above begins and ends with all worms in the population having the same allele.

The forces of evolution

In the above examples, the populations of worms are not evolving--neither the genotypes nor phenotypes are changing. For evolution to occur, there must be mutation, selection, or random genetic drift. These are the three major forces of evolution. The cause changes in genotypes and phenotypes over time. They also determine the amount and kind of variation seen in a population at a given time. This simulation focuses on drift (mutation and selection are covered in later simulations).

Random genetic drift

When genetic drift is introduced into the model, the results are different:

Note that in generation 2, the pink worm produces 1 offspring, the 3 green worms produced none, and the dark blue worm produced 4.

The role of chance

In real life, some individuals have more offspring than others--purely by chance. The survival and reproductions of organisms is subject to unpredictable accidents. It doesn't matter how good your driftworm genes are if you get squished by a shoe before producing offspring.

1. An ant gets stepped on.


2. A rabbit gets swept up by a tornado.


3. An elephant drinks up a protozoa living in a puddle.


4. A plane crashes killing a Nobel Laureate.

None of the above events has anything to do with the dead organism's genotype or phenotype these events occurred purely by chance.

Fixation of an allele

In a population model with genetic drift, alleles will eventually become "fixed". When an allele is fixed, all members of the population have that allele. In the graphic below, note that the dark blue allele fixed after 4 generations.

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Natural Selection

Natural selection is the process where heritable traits that make it more likely for an organism to survive long enough to reproduce become more common over successive generations of a population. It is a key mechanism of evolution. The natural variation within a population of animals, plants, bacteria, etc. means that some individuals will survive better than others in their current environment.

Fitness The concept of fitness is central to natural selection. Broadly, individuals which are more "fit" have better potential for survival, as in the well-known phrase survival of the fittest. Modern evolutionary theory defines fitness not by how long an organism lives, but by how successful it is at reproducing. If an organism lives half as long as others of its species, but has twice as many offspring surviving to adulthood, its genes will become more common in the adult population of the next generation. This is known as differential reproduction. Though natural selection acts on individuals, the effects of chance mean that fitness can only really be defined "on average" for the individuals within a population. The fitness of a particular genotype corresponds to the average effect on all individuals with that genotype.

Types of selection: The unit of selection can be the individual or it can be another level within the hierarchy of biological organisation, such as genes, cells, and kin groups.

Natural selection occurs at every life stage of an individual. An individual organism must survive until adulthood before it can reproduce, and selection of those that reach this stage is called viability selection.

In many species, adults must compete with each other for mates via sexual selection, and success in this competition determines who will parent the next generation.

When individuals can reproduce more than once, a longer survival in the reproductive phase increases the number of offspring, called survival selection.

The fecundity of both females and males (for example, giant sperm in certain species of Drosophila) can be limited via fecundity selection. The viability of produced gametes can differ, while intragenomic conflicts such as meiotic drive between the haploid gametes can result in gametic or genic selection.

The union of some combinations of eggs and sperm might be more compatible than others; this is termed compatibility selection.

It is also useful to distinguish between ecological selection and the narrower term sexual selection. Ecological selection covers any mechanism of selection as a result of the environment (including relatives, e.g. kin selection, competition, and infanticide), while sexual selection refers specifically to competition for mates. Sexual selection can be intrasexual, as in cases of competition among individuals of the same sex in a population, or intersexual, as in cases where one sex controls reproductive access by choosing among a population of available mates. Most commonly, intrasexual selection involves male-male competition and intersexual selection involves female choice of suitable males, due to the generally greater investment of resources for a female than a male in a single offspring organism.

PROTEINS

Proteins (also known as Polypeptides) are organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.

The sequence of amino acids in a protein is defined by the sequence of a gene, which is encoded in the genetic code. In general, the genetic code specifies 20 standard amino acids, however in certain organisms the genetic code can include selenocysteine – and in certain archaea – pyrrolysine. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism. Proteins also have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape.

Amino acids table:

Amino acid list:

Synthesis:

Protein structure:
Most proteins fold into unique 3-dimensional structures. The shape into which a protein naturally folds is known as its native conformation. Although many proteins can fold unassisted, simply through the chemical properties of their amino acids, others require the aid of molecular chaperones to fold into their native states. Biochemists often refer to four distinct aspects of a protein's structure:

Primary structure: the amino acid sequence.

Secondary structure: regularly repeating local structures stabilized by hydrogen bonds. The most common examples are the alpha helix and beta sheet. Because secondary structures are local, many regions of different secondary structure can be present in the same protein molecule.

Tertiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another. Tertiary structure is generally stabilized by nonlocal interactions, most commonly the formation of a hydrophobic core, but also through salt bridges, hydrogen bonds, disulfide bonds, and even post-translational modifications. The term "tertiary structure" is often used as synonymous with the term fold. The Tertiary structure is what controls the basic function of the protein.

Quaternary structure: the shape or structure that results from the interaction of more than one protein molecule, usually called protein subunits in this context, which function as part of the larger assembly or protein complex.

VIDEO SUMMARY:

http://www.youtube.com/watch?v=lijQ3a8yUYQ

DNA CELLULAR DYNAMICS: REPLICATION TRANSCRIPTION AND TRANSLATION

DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is "semiconservative" in that each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication. Please look this excellent video about DNA replication.

www.youtube.com/watch?v=teV62zrm2P0

Transcription is the synthesis of RNA under the direction of DNA. RNA synthesis, or transcription, is the process of transcribing DNA nucleotide sequence information into RNA sequence information. Both nucleic acid sequences use complementary language, and the information is simply transcribed, or copied, from one molecule to the other. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell.

www.youtube.com/watch?v=vJSmZ3DsntU

Translation is the first stage of protein biosynthesis (part of the overall process of gene expression). Translation is the production of proteins by decoding mRNA produced in transcription. Translation occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and large subunit which surrounds the mRNA. In translation, messenger RNA (mRNA) is decoded to produce a specific polypeptide according to the rules specified by the genetic code. This uses an mRNA sequence as a template to guide the synthesis of a chain of amino acids that form a protein.

www.youtube.com/watch?v=5bLEDd-PSTQ

Finally, it is very important that the student understand the connection between those three important processes for that reason look this video.

www.youtube.com/watch?v=NJxobgkPEAo&feature=related