BIOLOGY

• Biotechnology is the manipulation of organisms or their components to make useful products
• Genetic engineering is the direct manipulation of genes for practical purposes.
• DNA cloning is the process of making multiple, identical copies of a particular piece of DNA.
• Recombinant DNA (rDNA) is a molecule containing DNA from two different sources, very often different species.

   
• A.   PROCESS IN GENETIC ENGINEERING TECHNOLOGY

  1.   Identification and isolation of Gene of interest or DNA fragment to be cloned.

  2.   Insertion of isolated gene in a suitable vector.

  3.   Transformation is the introduction of vector into a suitable host.

  4.   Selection of the transformed host cell.

  5.   Multiplication or expression of the introduced gene in the host.

   
• RESTRICTION  ENZYME  is  a  DNA-

  cutting enzyme that recognizes a specific target sequence (restriction site) and cuts DNA into two pieces at or near that site.
• DNA LIGASES join together fragments of newly synthesized DNA to form a seamless strand
• A.   VECTORS

  ●         Cloning vector is a DNA molecule that can carry foreign DNA into a host cell and be replicated there.

  ○       Example: PBR322, PUC

   

  ●         PLASMID is a small, circular DNA molecule that is replicated separately.

  ○       Bacterial plasmids are widely used as cloning vectors.

   
• 
  

  A. PROCESS OF BUILDING A RECOMBINANT PLASMID

  1. IDENTIFICATION AND ISOLATION

  2. Insertion / ligation

  3. transformation

  4. selection

  5. Multiplication
• 1. Identification

  • Start with a target gene and a circular plasmid
  • The target gene has two EcoRI restriction sites near its ends
  • The plasmid has one EcoRI site after a promoter that drives expression in bacteria
  • EcoRI sites sequence: 5'-GAATTC-3' 3'-CTTAAG-5
• 1. Isolation:

  • Use EcoRI to separately digest the gene fragment and the plasmid
  • All ends have an overhang of four nucleotides, known as sticky ends
  • EcoRI's cut pattern:

  5'-G|AATTC-3' 3'-CTTAA|G-5'
• 2.  INSERTION

  ●         Take the gene fragment and the linearized (opened-up) plasmid and combine them along with DNA ligase.

  ●         The sticky ends of the two fragments stick together by complementary base pairing.

  ●         However, there are still gaps in the sugar-phosphate backbones of the DNA double helix at the junction sites where the gene and plasmid DNA meet.
• 2. LIGATION

  ●         The use of ligase joins the fragments together.

   

  Once they are joined by ligase, the fragments become a single piece of unbroken DNA. The target gene has now been inserted into the plasmid, making a recombinant plasmid.
• 3.   TRANSFORMATION is the transfer of DNA into bacteria. 4.   SELECTION The use of antibiotic selection and DNA analysis methods to identify bacteria that contain the plasmid we’re looking for.

   
• Steps of bacterial transformation and selection:
• 1. Specially prepared bacteria are mixed with DNA (e.g., from a ligation)
• 2. The bacteria are given a heat shock, causing some of them to take up a plasmid
• 3. Plasmids used in cloning contain an antibiotic resistance gene. All bacteria are placed on an antibiotic plate to select for those that took up a plasmid
• 4. Bacteria without a plasmid die, and each bacterium with a plasmid forms a cluster of identical, plasmid-containing bacteria called a colony
• 5. Several colonies are checked to identify one with the correct plasmid (e.g., by PCR or restriction digest)
• 6. A colony containing the correct plasmid is grown in bulk and used for plasmid or protein production
• 5.   MULTIPLICATION

  A.   Bacteria as plasmid factories

  ●  Bacteria can be simply used as "plasmid factories," making lots of plasmid DNA that might be used in further DNA cloning steps (e.g., to build more complex plasmids) or in various types of experiments.

  
  

  B.   Bacteria protein factories

  ●  Bacteria may also be used as protein factories where they are induced to express the gene it contains and lysed (split open) to release the protein
• DNA DEOXYRIBONUCLEIC ACID carries the genetic information in all living organisms
• RNA RIBONUCLEIC ACID a single stranded type of nucleic acids that is mostly involved in protein synthesis. Enzyme is a protein molecule that catalyzes biochemical reaction.
• Genetic Engineering -the artificial manipulation, modification, and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organisms
• Classical Breeding -the practice of mating or breeding selected organisms with desirable traits
• 1. Steps in Classical BreedingDetermine which trait are significant enough to be chosen.2. Select parents that exemplify these traits.3. Choose the best offspring from parents to produce the next generation.4. Repeat the process
• 1. Genetic Engineering The modification in genetic engineering may involve the following:introduction of new traits into an organism,2. enhancement of a present trait by increasing the expression of the desired gene, or3. enhancement of a present trait by disrupting the inhibition of the desired genes' expression
• Geologic Time Scale - a record of life forms and geological events in Earth's historyFossils -remnant, impression, or trace of an animal or plant of a past geologic age that has been preserved in Earth's crustThe complex of data recorded in fossils worldwide - known as the fossil record - is the primary source of information about the history life on Earth
• EON - the largest division of geologic time scale; half billion - nearly 2 billions of yearsPRECAMBRIAN - 4.5 million years; about 88% of the earth's historyHadeasArcheanProterozoic
• ERA -divisions that span time periods of tens to hundreds of millions of yearsPaleozoic EraMesozoic EraCenozoic Era
• GMO Genetically Modified Organisms = final product that undergo genetic engineering
• Vectors = medium
• Plasmid = small, circular DNA molecule that is replecated separately
• Applications of rDNA:

  1. Agriculture
  2. Bioremediation
  3. Pharmaceuticals
  4. Genetic Testing
  5. Genetic Therapy
• rDNA Health Effects:

  1. Allergic Reactions
  2. Cancer Formation
  3. Loss of Nutrition
  4. Toxic Effects
  5. Compromised Immunity
• rDNA in Biodiversity:

  1. Horizontal Gene Transfer
  2. Invasion of GMOs in Wild
  3. Decline in Biodiversity
  4. Reduction in Variation
  5. Loss of Wild Species
• rDNA in Ethics:

  1. Playing God argument
  2. Going against what is natural
  3. Possible depression
  4. Reduction in Variation
  5. Corporate Exploitation
• Why say no to rDNA:

  1. Possible Health Effect
  2. Biodiversity
  3. Ethics