Construction of a recombinant DNA

Construction of a recombinant DNA
DNA cloning. Vectors (plasmids)
Genetic engineering: agriculture and environment
genetic engineering and medicine
Genetic engineering is a set of techniques that allow manipulation and transfer of genes from one organism to another. Thus we obtain genetically modified organisms. Transgenic organisms are those which has been inserted a gene, known as transgene from another organism.
DNA formed by joining DNA fragments from different organisms is called recombinant DNA.
CONSTRUCTION OF A RECOMBINANT DNA
The basic tools for the construction of recombinant DNA molecules are restriction endonucleases and DNA ligases.
The DNA can be cut into fragments by enzymes called restriction endonucleases. These DNA recognize specific frequencies and cut in specific places, so that the resulting fragments are (complementary) cohesive edges to other ends of DNA cut with the same enzyme.
The DNA ligase are enzymes that bind the cohesive ends of DNA fragments generated by restriction endonucleases. And they can be cut and join DNA fragments of different origin, thus it is possible to introduce a DNA into DNA of organisms other. The restriction enzymes are the scissors of DNA and DNA ligase the glue that binds the cut edges with scissors. There are more than one restriction enzyme and each recognizes and cuts a particular sequence in DNA.


Seen in the drawing on the right as a recombinant DNA is obtained:
First DNA from two different organisms is cut with EcoRI restriction enzyme. This enzyme recognizes the sequence GAATTC and short between the G and A of each of the DNA molecules nucleotides.
Second DNA fragments with complementary cohesive obtained in each of the molecules.
Third are contacted DNA fragments obtained and subjected to DNA ligase enzyme, forming a recombinant DNA molecule containing DNA of the two organisms.
DNA cloning. Vectors (plasmids)
DNA cloning is possible by two methods:
Introducing the DNA fragment is to multiply in a bacterium, the bacterium also multiply the multiplied DNA (cloning).
By the PCR DNA you get copies you want quickly and easily.
The PCR (polymerase chain reaction) technique allows many copies of DNA fragments in a short time and starting from a very small amount of DNA. To do this we introduce the DNA fragment that we want to copy in a liquid containing the DNA polymerase enzyme and many nucleotides. First it is heated to separate the two strands of DNA, once separated DNA polymerase (DNA polymerase is not denatured by heat, because it has been obtained from a thermophilic bacterium that lives in places with high temperatures) will placing the complementary nucleotides making copy, allowed to cool to rejoining the DNA strands and the process is repeated every time you want (each cycle is multiplied exponentially DNA copies obtained).
In the PAU only enters the first type of DNA cloning.
The cloning of a gene is introducing into a cell so that it can be copied and maintained. For this, the gene is inserted into a DNA molecule, called cloning vector, capable of entering and replicating independently in a host cell.
There are various types of cloning vectors, but one of the most used are plasmids, which are small circular DNA molecules that can replicate independently (ie, without associating the chromosomal DNA) in bacteria. Plasmids containing the inserted DNA fragment are called recombinant plasmids.
If a recombinant plasmid is introduced into a bacterium it will be replicated with it and obtain copies of that million recombinant plasmid can be isolated. The process of cloning a gene in bacteria includes the following steps:
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1- Preparation of DNA fragment containing the gene to be cloned.
2- Preparation of recombinant plasmid: To insert the gene to be cloned into the cloning vector (plasmid in this case), the plasmid and the DNA to be cloned with the same restriction enzyme and bind both cut with DNA ligase to obtain the recombinant plasmid. In addition to the gene we want to clone, the plasmid carries a gene called easy genotypic detection marker, needed to step 4.
3- Transformation of bacteria: consists of incorporating the recombinant plasmid in bacteria, for this purpose, recombinant plasmids were incubated in a culture of bacteria able to capture foreign DNA, a process called transformation (remember the parasexualidad in bacteria: conjugation, transformation and transduction).
4- Selection of the transformed bacteria: consists of checking that bacteria have incorporated the gene. As the plasmid carries an easy marker to detect gene, for example resistance to an antibiotic (other may be producing bioluminescence but bacteria almost always an antibiotic resistance is used), then added to the bacterial culture medium that antibiotic and bacteria that survive are those that have incorporated the recombinant plasmid, and therefore we want to clone the gene.
5- growth of transformed bacteria: The transformed bacteria are maintained in culture media for rapid growth. While bacteria are doubled, the number of recombinant plasmids and bearing inserted gene is also doubled.
6- Isolation of recombinant plasmids and copies of the gene of interest.
The cloning of clones obtained is called library or genomic DNA library.
GENETIC ENGINEERING: AGRICULTURE AND ENVIRONMENT
Let's see the process more used to obtain transgenic plants and applications of genetic engineering in agriculture and the environment.
Production of transgenic plants: Transformation (Agrobacterium) and Regeneration
A transgenic plant is a plant whose genome has been genetically engineered either to introduce one or more new or to modify the function of a separate gene.
The most common method to introduce genes into plants using a soil bacterium called Agrobacterium, which under natural conditions is capable of infecting plant cells and transferring genes (under natural conditions producing plant disease infects "crown gall"). This bacterium has a plasmid called Ti plasmid, which is used as a cloning vector in plants.
During infection the bacteria transfers a fragment of the Ti plasmid to the plant cells. This fragment is called T-DNA integrated and ends somewhere in the chromosome.
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The production of transgenic plants consists of two stages called transformation and regeneration. Transformation is the process of integration into the plant cell the gene or genes that interests us and regeneration consists in obtaining a whole plant from a transformed plant cell.
Transformation: Genetic engineering can insert a gene of interest in the T-region of the Ti plasmid. For this, the Agrobacterium Ti plasmid, is isolated, inserted into the T region of the plasmid the gene of interest, using a restriction enzyme and DNA ligase. It reintroduces Agrobacterium plasmid in bacteria and allowed to infect the bacteria plant cells Thus, after infection, the new gene will also be transferred to the plant cell and inserted into the plant genome. Not all species can be transformed using Agrobacterium. Especially for monocots it has developed an alternative method, known as "microparticle bombardment". In this method, microparticles of gold or tungsten with DNA, which are accelerated in a "gene gun" to gain enough speed and power to penetrate the cell lining.
Feedback: After transformation, plant cells receiving the genes of interest are selected using antibiotics or herbicides in the growth medium (plus the genes of interest, other genes, denominated "selectable markers" which confer resistance cells that carry them, so that cells do not carry these marker genes, die). Plant cells that have survived are those that have undergone transformation. Then they are grown and complete plants (regeneration) are obtained. Much of the plant cells are totipotent, meaning that a cell from any part of the plant can multiply and generate the entire plant. For that cells grow in the appropriate culture medium and in the presence of certain hormones and plant factors. The result is a plant carrying the gene of interest in each of their cells.
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Obtaining plants resistant to the herbicide glyphosate: In the image on the left shows that glyphosate inhibits an enzyme (epsps) vital to the survival of plants, causing their death. Glyphosate tolerant plants have a variant epsps gene of strain CP4 the soil bacterium Agrobacterium tumefaciens.que produces a epsps enzyme is not inhibited by glyphosate.
As the EPSPS enzyme produced in this bacterial strain is not affected by glyphosate, its introduction into the genome of plants tolerant to the herbicide becomes. The introduction of this gene has been made by the technique described above: the epsps gene is introduced into the T-region plasmid of Agrobacterium, and plant cells are infected with the Agrobacterium bacterium having the recombinant plasmid with epsps gene resistant to the herbicide, some plant cells will be transformed and possess the gene which gives resistance to the herbicide glyphosate. To select these plant cells glyphosate herbicide and plant cells are transformed survive added. These cells whole plants (regeneration) glyphosate resistant transgenic be obtained.
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Applications of transgenic plants in agriculture
The objectives in agriculture is to introduce genes making transgenic plants generally more productive. For example plants that give larger tomatoes, in as much or more tasty. Other objectives are to achieve herbicide-resistant plants, drought and disease or get more nutritious fruit content (for example vitamins). Also it is investigated using transgenic plants to produce human proteins as drug substances for medical use or viral proteins for use as a vaccine.
Environmental applications of genetic engineering
Some microorganisms and very few plants are widely used to remove contaminants, there are species that degrade oil and pesticides or heavy metals accumulate or decontaminated wastewater. The genes of organisms involved in these processes can be introduced into other organisms. This way could eliminate oil spills, toxic spills or contamination.
The use of living organisms, mainly bacteria to eliminate environmental pollution is called bioremediation. The most famous is the removal of oil slicks using bacteria capable of digesting petroleum hydrocarbons transforming them into less or no polluting substances. In the same way, they have created bacteria capable of living in the presence of heavy metals and remove them from ecosystems through various chemical reactions. Also biodegradable plastics can be produced and degraded by certain bacteria ??
GENETIC ENGINEERING AND MEDICINE
Applications of genetic engineering in medicine
Some people do not make certain proteins essential for life as insulin, growth hormone, clotting factors ??
The genes needed to make these proteins can be introduced into microorganisms such as bacteria that manufactured these proteins to give to sick people. These microorganisms produce large quantities at low prices and are risk-free human proteins not as was formerly that of animals and had pulled disease risk and side effects.
The coordinator sent us this selectivity is the same: Getting pharmaceuticals (taken from the book Biology 2 Baccalaureate Editorial SM)
Many diseases are caused by the lack of a protein: Insulin, interferon, growth hormone or factor VIII coagulation are proteins produced in very small amounts by expensive processes and that, at present, are manufactured by genetic engineering.
You can also introduce genes into bacteria for producing other drugs such as antibiotics, vaccines ?? For example we get a virus gene that makes the virus antigen, is we put a bacterium and the bacterium produces antigens without the virus that are used as vaccine. Currently, it is being investigated introducing human genes in the pig to make organs that can be transplanted into humans with the least possible rejection.
Gene therapy is either eliminate the abnormal gene that causes a disease, or by entering the (correct) no abnormal gene to prevent the disease. In the case of many genetic diseases they could be avoided with gene therapy but is currently under investigation.
Obtaining insulin
Insulin was the first genetically engineered protein. It consists of two polypeptides, the A and B, and their production is necessary first chemically synthesizing two DNA strands which express: the A chain and the chain. Genes are inserted separately, and together with the gene that expresses a protein -the ß-galactosidasa- in E. coli plasmids, recombinant plasmids were obtained.
These are introduced in different strains of E. coli, which are expressed and a fusion protein of-the-Gal ß-insulin-, which is more stable in E. coli insulin alone is obtained. These fusion proteins are chemically processed to separate them polypeptides A and B, then, in the laboratory, by renaturation and oxidation of the cysteines, are joined to obtain active insulin.