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Genetic Engineering General Steps

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    GENETIC ENGINEERING GENERAL STEPS
    What is Genetic Engineering?
    Genetic Engineering – inserting a foreign gene of interest into a host to transcribe and translate a particular protein. Ex. Inserting the human insulin gene into bacteria to mass produce it. General Steps

    Obtain the gene of interest (ex. insulin gene)
    Insert the gene into the host (ex. bacteria)
    Allow the host to multiply and express the foreign gene
    get your desired protein!
    Get lots of cells that can make the protein = clones

    The Big Picture
    The inserted gene is transcribed and translated using the RNA Polymerase, ribosomes and other resources in the cell Plasmids
    Circular DNA
    Extrachromosomal
    NOT part of the E. coli genome
    “extra” DNA
    Contain a few non-essential genes
    Can give the bacteria additional “traits”
    Depends on the genes on the plasmid
    Can be exchanged between bacteria
    Recombinant plasmids
    Plasmids can be modified in biological labs
    Modified plasmid = Recombinant plasmid
    Plasmids can be used as cloning vectors to get the recombinant plasmid into E. coli Cloning vectors = way to get the gene of interest into the host Transformation
    Process in which foreign DNA is physically inserted into host E. coli cells. E. coli that contains recombinant plasmid = Transformed cell

    Transformation Steps
    Recombinant plasmids and host E. coli are mixed together
    CaCl2 is added
    The Ca2+ ions neutralize the negative charges on plasmid DNA Help plasmid enter the membrane
    Transformation Steps
    Heat Shock
    By rapidly changing the temperature of the solution, temporary pores are opened in the membrane Creates an opening for the plasmids to enter the E. coli
    Transformation
    Transformation is not 100% successful
    After transformation
    Some cells will contain plasmid = transformed
    Some cells won’t contain plasmid = untransformed
    In a later step, you will determine which cells were transformed E. Coli as a host
    E. coli is a good host because:
    Reproduce quickly (once every 20 minutes)
    Nonpathogenic (the strain we use is not harmful)
    Genome fully characterized (all genes have been sequenced)
    HISTORY OF
    GENETIC ENGINEERING

    The beginnings
    1869
    DNA isolated
    1944
    DNA proved to be the hereditary material
    1953
    DNA structure determined
    Working with DNA
    1961
    DNA can be split on heating and stuck back together again on cooling (reannealing) DNA hybridisation possible
    1962
    The first restriction endonucleases discovered
    Molecular scissors
    Working with DNA
    1966
    The genetic code worked out
    Discovered to be a universal code
    1967
    DNA ligase discovered
    Molecular glue
    Fragments of DNA can be stuck together
    Recombinant DNA technology
    1973
    DNA cloning carried out on bacteria
    Gene identified
    Cut with restriction enzyme
    Spliced into a plasmid using ligase
    Plasmid reintroduced into a bacterium
    Gene copied whenever the bacterium divides
    Non-bacterial gene can be expressed in the bacterium
    Recombinant DNA technology
    1977
    Rapid sequencing of DNA developed
    1982
    Transgenic mice and fruit flies produced
    Recombinant DNA technology
    1983 Polymerase chain reaction invented
    1985 Genetic fingerprinting developed
    Medicine
    1989
    Cystic fibrosis gene cloned and sequenced
    1990
    Trials for gene therapy begin
    Agriculture
    1994
    Genetically modified organisms for food
    Human Genome Project
    1990-2003The human genome worked out
    Goals of the human genome project
    identify all the approximately 20,000-25,000 genes in human DNA determine the sequences of the 3 billion chemical base pairs that make up human DNA store this information in databases
    improve tools for data analysis
    transfer related technologies to the private sector
    address the ethical, legal, and social issues (ELSI) that may arise from the project Genomics
    Today:
    Over 70 Eukaryotes sequenced
    About 25 of these are mammals
    Over 350 prokaryotes have been sequenced
    FACTS ABOUT GENETIC ENGINEERING
    Definition of GE
    The technology entailing all processes of altering the genetic material of a cell to make it capable of performing the desired functions, such as producing novel substances. Facts about GE
    The result is called genetically modified organism(GMO)
    The Major aim is to produce much food at low cost to reduce the world hunger Can potentially be used in humans to change their appearance, intelligence, character and adaptability Facts about GE
    The new gene technologies enable creating life forms never existed before Strategies are to feed GMO to humans as well as to fishes and livestock that on the other hand can be fed to humans. Pros & Cons of GE

    Is GE precise enough ?
    Pro arguments:
    Scientists use „gene guns“ to insert the specific gene in the organism precisely Contra arguments:
    The choice of gene is precise. But the insertion of this gene into a living cell is imprecise. There is no control where in the DNA the new gene is inserted. This process can disrupt the DNA Pros & Cons of GE

    Effects on the environment
    Pro arguments:
    GE minimizes soil erosion by reducing the need of flowing . Plants resisant to weather , climate insect infestation, desease, molds and fungi. Contra arguments:
    GE can create toxins, noxious-vegetation, harm to wild life and may create new molds and fungis. Pros & Cons of GE
    Effects on the environment
    Pro arguments:
    GE allows the creation of thousands of novel life forms in a brief moment. Contra arguments:
    Once GMOs are released into the environment they cannot be recalled therefore they are a very dangerous kind of pollution. Pros & Cons of GE
    Effects on warfare
    Pro arguments:
    Biological weapons can determine a war.
    Contra arguments:
    GE could be used to create biological weapons.
    Pros & Cons of GE
    Effects on the agriculture
    Pro arguments:
    Farmers can spray in order to kill weeds without killing the crops. Contra arguments:
    Furthermore the weeds might develop their spray resistance and greater herbicide resistance has to be created. The virus-resistance might also create new viruses that never existed before.

    Pros & Cons in general
    Genetic engineering reduces costs of production This means that the poor can afford more food Cheaper and safer source of human medicine
    Higher productivity
    GE can reduce the World hunger
    GE is unnatural
    GE crosses species barriers which would never occur in nature A high danger might be the “gene-flow”-transfer of genes from crops to weedy relatives by cross-pollination

    Pros & Cons in general
    There is also the fear of what will happen with biotech industry if the genetically engineered organisms cause a environmental catastrophe, which will be followed by costly damage to flora and fauna The healthiness of the humans is threatened by the genetically engineered food because it is not sure if there will be allergical reactions President Bush

    George Bush is for a total ban of GE or human cloning
    „Our children are not product to be designed and manufactured“ Against reproduction of stem cells

    BASIC STEPS IN
    GENETIC ENGINEERING
    What is genetic engineering
    Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all. Basic steps in genetic engineering

    Isolate the gene
    Insert it in a host using a vector
    Produce as many copies of the host as possible
    Separate and purify the product of the gene
    Step 1: Isolating the gene
    Step 1: Alternative method (using reverse transcriptase)
    Reverse transcriptase
    mRNA converted into cDNA
    Complementary strand produced using DNA polymerase
    Advantage – more mRNA in cell than DNA
    Step 2: Inserting gene into vector
    Vector – molecule of DNA which is used to carry a foreign gene into a host
    cell

    Step 3: Inserting vector into host
    See worksheet
    Replica plating
    Step 4: Multiplication of the host cells by cloning
    Large scale fermenters by cloning
    All genetically identical because of asexual reproduction

    PRINCIPLES OF
    GENETIC ENGINEERING

    TYPES OF GENETIC ENGINEERING
    Types of Genetic Engineering
    Negative
    Positive
    Negative Genetic Engineering
    Correct a genetic defect
    Positive Genetic Engineering
    Make a life-form better.
    Types of Genetic Therapy
    Somatic Therapy
    Germ line Therapy
    Splicing
    Splicing is a method where genes from one organism are “spliced” into the DNA of another organism. This is the most common method of genetic engineering. Genetic Supermarket
    In the genetic supermarket parents would have a limited voice in opting for genetic engineering of both positive and negative features. The government would simply ensure that no dangerous modifications were made. Cloning

    Evolution and Science
    Many feel that evolution is wrong or false.
    Science, such a genetics, helps to confirm evolution of life on our planet on a daily basis. Evolution
    Darwin’s Theory of Natural Selection, Evolution of Species.

    Charles Darwin Published the Origin of Species in 1859
    Dolly the Sheep
    In 1997 Scientist in Scotland cloned the first mammal- Dolly the sheep. Not everyone was pleased.

    In 2003 Dolly passed away from complications related to her cloning. Splicing in Plants
    Glowing plants

    USDA Organic
    The USDA now certifies all food that has an organic label to be tested. Cheese
    More than 70% of all cheese sold in the US is made with Genetically Modified enzymes GE papaya
    Farmers in the orient ganble that GE papaya will have a longer shelf life and surive the trip to the US and Europe. Soybean
    60% of the US soybean crop is genetically modified.
    Canola Oil
    62 % of the plants from which canola oil is extracted are genetically modified. Vitamins and Minerals
    Genetically Modified bacteria and yeast have been used since 1981 to produce vitamins and nutritional supplements. Many pharmaceutical drugs are the result of genetic engineering. 70% of all processed food

    70% of all processed food is the United states contains Genetically Modified Components. CONCLUSION
    Genetic engineering can cure diseases and improve upon existing plants and animals.

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