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					Biotechnology
Reading quiz
 l   Identify the term that best represents each
     description
     l   1. When a bacteria is not affected by chemicals that
         interfere with its life processes
     l   2. A rod shaped bacterial cell
     l   3. Chemicals that interfere with bacteria’s life
         processes
     l   4. Substance discovered by Alexander Fleming that
         can be used to treat bacterial diseases
 l   5. Draw a picture of a typical bacterium and
     label with cell wall, pili, nucleoid region
     (genetic material)
What Are Common Uses of
Biotechnology?
 l Identification   (“DNA fingerprinting”)
 l Diagnosing    and treating genetic
 l diseases  modifying organisms to
   Genetically
   make medicines or other useful
   products
   - Human Growth Hormone
   - Rice with added vitamins and
   minerals for use in third-world countries
  - Human tissues grown in animal
  donors
 Your Health
• Genetically     l   Making new or cheaper• Testing
  Modified            drugs
                                                  for and
                      l   Insulin from bacteria
  Organisms                                       treating
                      l   Antibiotics in milk
  – Pesticide                                     genetic
    producing                                     diseases
    crops                                         – Cancer
  – Human                                         – SCIDS
    organs in
    animals for
    transplants
Genetic Engineering
 l Selective breeding
   l Advantages?
   l Disadvantages?
 l Increasing variation
 l Induce mutations
This is the Biotechnology Age

You have been born in it and will live during it


You must be informed in order to make
intelligent decisions


And… you deserve to be qualified to work in an
exciting field - as scientists, technicians, receptionists,
business execs, and artists
 Prokaryotes are identified by their shape, chemical
 nature of cell walls, movement, and how they obtain
 energy.

     SHAPE:
     Rod- bacilli
     Spiral- spirilla
     Spherical- cocci
CELL WALLS: Scientists use Gram
staining to tell cell wall type apart.
         If the Gram testing is
positive, then the bacteria has thick
peptidoglycan walls
         If the Gram testing is
negative, the bacteria has thin walls
inside an outer lipid layers.
MOVEMENT: Flagella.
        Metabolic Diversity
   Identifies prokaryotes by how they obtain energy.



Heterotrophs
Chemoheterotrophs- consume organic molecules for energy and a
  supply of carbon.
Photoheterotrophs- use photosynthesis; need organic compound like a
  carbon source.

Autotrophs
Photoautotrophs- use photosynthesis EX: cyanobacteria-
Chemoautotrophs- make organic carbon molecules from CO2. They
  obtain energy directly from chemical reactions.

Releasing energy- by fermentation, cellular respiration, or both
Obligate aerobes- constantly need oxygen
Obligate anaerobes- live without oxgen
Facultative anaerobes- live with/without oxgen EX: E. coli
     Life Cycle
Bacteria can divide at high speeds, (every 20 min. )


Binary Fission- (asexual).

Conjugation- Bacteria exchange genetic info by
 forming a bridge between two of them. Different
 genes are transferred to each other to increase
 genetic diversity in bacteria populations.

Transformation-To be discussed later

Spore formation
Antibiotics and resistance

 l Alexander     Fleming and Penicillin
 l Antibiotics
 l Antibiotic   resistance
Bacteria’s impact on life

 l Pathogenic   bacteria
 l Toxins
 l Biowarfare
 l Production  of chemicals and foods
 l Mining, environmental use
 l Nitrogen fixation
 l Decomposers
What Are The Basic Tools Of
Biotechnology?

    Micropipets



    Gel boxes
     Micropipets
l   Used for accurately measuring very
    small amounts of fluids
l   Used in almost all biotechnology to
    measure
l   1 μL = .001 mL = .000001 L = 1 x
    10^-6 L
l   2 mL = μ L = L
                               Pipet types
       P-20 (2-20 μ L)   P-200 (20-200 μ L)   P-1000 (200-1000 μ L)
Reading quiz

 l Viruses contain a coat made up of ____
   and their genetic material is _____
 l Lytic and lysogenic are examples of
   what?
 l How is HIV different than many other
   viruses?
The problem…
l   A disease is causing large pale
    spots on the leaves of tobacco
    plants
l   1892, Ivanovski identified the
    cause of the disease to be in the
    juice
l   1897, Beijerinck suggested that
    tiny particles caused the
    disease, and named them
    viruses (Latin-poison)
Viral structure
Life cycles
HIV and AIDS

 l   HIV is the virus that can lead to AIDS
 l   Transmission
 l   Prevention
     l   AVOID any of the forms of transmission!
l   HIV mutates, evolves very rapidly
l   Evades defenses of the immune system
l   Destroys Helper T cells
Is it living?

  l Contains   genetic material? YEP
  l Evolves? YEP
  l Interacts with other organisms? YEP
    (parasitic)
  l Metabolic activity? IN A HOST CELL
  l Reproduces? IN A HOST CELL
  l What do you think?
Reading quiz (get SG 3,4C)


  l   Write the term that best represents each
      description
      l   1. Circular piece of DNA that replicates
          independently of the chromosome(s)
      l   2. Protein that can “cut” DNA at precise spots
      l   3. Purposeful manipulation of DNA
      l   4. Overhanging sequence of bases left after DNA is
          “cut”
      l   5. Substance that can be used to identify cells with
          our gene of interest
1 point each
   l   1. Circular piece of        l   Plasmid (vector okay)
       DNA that replicates
       independently of the
       chromosome(s)
   l   2. Protein that can “cut”   l   Restriction enzyme
       DNA at precise spots
   l   3. Purposeful               l   Genetic engineering
       manipulation of DNA
   l   4. Overhanging
       sequence of bases left      l   Sticky end
       after DNA is “cut”
   l   5. Substance that can       l   Antibiotic (tetracycline
       be used to identify cells       okay)
       with our gene of
       interest
    Genetic engineering defined:

Recombination of genetic information by
transferring genetic information from one
organism
to another, usually by way of a recombinant
“vector”


    At a minimum from different species --
          from one bacterial species to another...

    Or… as dramatic as DNA from different kingdoms!

 Bacteria                     Plant                  Animal
Uses in basic research
Simple:
- to produce multiple copies of DNA

- to produce multiple copies of a
   protein


More complex:

 - monitor spread of virus
   infecting tobacco plants
Transgenic organisms
l   Organisms contain
    genes from other
    organisms
    l   Microorganisms
    l   Animals
    l   Plants
Tools to accomplish genetic engineering

  l Restriction   Enzymes
    l   Protein molecules used to cut DNA
  l Ligase   Enzymes
    l   Protein molecules used to put together DNA
  l Plasmids
    l   Small DNA molecules used to put new
        genes into bacteria
  l Viruses
    l   Used to put new genes into other living
        things
                how is DnA cut?


l   DNA is cut using restriction enzymes.
    l Examples for different restriction enzymes:

      l Eco RI, Hind III

l   Restriction enzymes cut DNA at specific four or six-
    base-pair-sequences called restriction sites.
    l Examples for different restriction sites:

      l Eco RI at G AATT C

      l             C TTAA G
      l Hind III at A AGCT T

      l             T TCGA A
How is a gene inserted?
Gene therapy
l   1. Identify gene
l   2. Clone gene
l   3. Replace the
    faulty or missing
    gene
l   4. Result? The
    correct protein
    should eliminate
    cause of the disorder
    l   Advantages
    l   Disadvantages
Transformation

          experiment
 l Griffith
 l Outside DNA gets incorporated into
   bacteria
 l What you have: pGLO plasmid (outside
   DNA) and E. Coli (bacteria)
Transformation procedure

l   How do you get       l   Compentency
    pGLO into E. Coli?       l   CaCl2
                             l   Heat/ice shock
l   How do you grow l Spread E. Coli on
    transformed E. Coli? petri dish with agar
                          and incubate
l   How do you identify l Amp resistance
    transformed cells? l Arabinose
                        l GFP protein
A plasmid used as
a vector must
haveorigin of replication
a. An                          Origin of plasmid              ON/OF
                               replication                    F switch
b. A gene conferring
resistance to an antibiotic.                          ar
                                                         aC




                                     i
                                    or
In addition, pGLO
contains                                       pGLO
c. The Green Fluorescent




                                                              G
Protein - the protein of




                                                             FP
                                      a l
interest                               b
d. An ON/OFF switch that
controls the synthesis of GFP Beta-                      Green
by the presence or absence of lactamase to               Fluoresce
the sugar, arabinose.         break down                 nt Protein
                              ampicillin
Tranformation prelab

 l   Read thoroughly through WB 204-206
 l   Make notes on any procedures you don’t
     understand. Highlight important information
 l   Use the background information to answer the
     following
     l   What is the role of the following items in the
         transformation lab and identify each as a gene,
         protein, or other: Ampicillin, GFP, Ampicillin
         resistant gene, arabinose, pGLO plasmid, E. Coli,
Interpreting results

 l   LB + DNA :
     Prediction, why?
 l   LB + DNA + amp :
     Prediction, why?
 l   LB + DNA + amp +
     arabinose :
     Prediction, why?
 l   Satellite colonies
Lab report
 l   Introduction
     l   Purpose
     l   Deduction
     l   Background info
 l   Materials/procedures
     l   Include amounts
     l   SUMMARIZE procedures (don’t copy from lab)
 l   Results
     l   Data table, picture, bar graph?, written paragraph
 l   Analysis
     l   Support/doesn’t support hypothesis? Interpret results from
         each petri dish. Difference between your results and class
         average? How can you improve the lab? Importance of lab?
Reciprocal teaching: “Are viruses
alive”
 l   As we’ve done before, take turns reading through the
     article (rotate after each paragraph). Each person will
     have a different role
     l   Summarizer: Summarizes EACH paragraph read
     l   Predictor: Predict what will happen next
     l   Connector: Discusses connections with class
         content/experiences/society
     l   Questioner: Asks under and on the surface questions. Asks if
         anyone else has questions

 l   Have your assignment sheet out. I will stamp WB 206
     (transformation activity sheet) and give you a stamp
     for your RT read based on your discussion
Reading quiz (get SG and transformation activity
stamped, and take out 207)

  l Write the term the best represents each
    description
     l 1. A weakened or killed pathogen
     l 2. A disease when the body launches an
       immune response against its own cells
     l 3. The first one was produced by Jenner
       using cowpox
     l 4. The body system HIV attacks
Antigen vs. antibody
Vaccines
l   A weakened or killed virus is injected into the
    individual
l   Elicits an immune response (antibodies)
l   Does not harm the individual
Gel Boxes

 l Used   to separate
   chemicals like DNA
   based on size,
   charge, etc..
 l Used for
   identification/DNA
   fingerprinting,
   researching genes,
   etc..
Lab B analysis
 l   Voltage vs. current
 l   When was electricity
     being conducted? Why?
 l   Electricity allows
     electrolysis to occur
 l   Role of phenol red?
 l   Role of buffer?
 l   Requirements for running
     DNA through a gel:
     Current and a stable pH
                      Note bubbles and swirls
                      of changing pH



H2O + NaCl + phenol red + TAE buffer
Number   Dye                Charge
1        Brilliant green
2        Janus Green B
3        Methylene Violet
4        Methylene Blue
5        Safranin O
6        Methyl Orange
7        Phenol Red
8        Bromophenol Blue
9        Loading Dye
10       Xylene Cyanol
the principles of Gel
electrophoresis
 l   Charge of DNA: ____
 l   If put into an electric field, it will move from the _____ to the
     ____ pole.
 l   A gel serves as the porous matrix for the movement of the DNA
     molecule.
 l   DNA is cut into fragments of different size.
DNA GEL
RFLP (DNA FINGERPRINTING)




                            libproto
Protocol
Polymerase Chain Reaction (PCR)

l   Purpose: Amplify DNA
    quickly
l   Applications: Acquire
    enough DNA to run a DNA
    fingerprint
l   Uses taq polymerase
Mammalian cloning
l   Nucleus of an
    organism’s egg is
    destroyed (lets say a
    sheep)
l   Nucleus is removed
    of a donor organism
    of the same species
    and put into the egg
l   That egg then
    develops into a clone
    of the donor’s DNA
Issues about cloning
 l   Cloning already happens in nature
     (identical twins, bacteria, asexual
     reproduction)
 l   What are consequences of
     mammalian cloning?
 l   How easy is it to do? Cloning the
     sheep Dolly took 277 attempts.
     What happens to unsuccessful
     eggs?
 l   Currently, there is a ban on human
     cloning

				
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posted:6/23/2013
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