Biotechnology

Reading quiz

Identify the term that best represents each description 1. When a bacteria is not affected by chemicals that

interfere with its life processes 2. A rod shaped bacterial cell 3. Chemicals that interfere with bacteria’s life

processes 4. Substance discovered by Alexander Fleming that

can be used to treat bacterial diseases 5. Draw a picture of a typical bacterium and

label with cell wall, pili, nucleoid region (genetic material)

Identification (“DNA fingerprinting”)Diagnosing and treating genetic diseasesGenetically modifying organisms to make medicines or other useful

products

What Are Common Uses of Biotechnology?

- Human Growth Hormone

- Rice with added vitamins and minerals for use in third-world countries

- Human tissues grown in animal donors

• Genetically Modified Organisms– Pesticide

producing

crops– Human

organs in

animals for

transplants

Your Health

Making new or cheaper drugs Insulin from bacteria Antibiotics in milk

• Testing for and treating genetic diseases– Cancer– SCIDS

Genetic Engineering

Selective breeding Advantages?

Disadvantages?

Increasing variation 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 makeintelligent 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- bacilliSpiral- spirillaSpherical- 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 DiversityIdentifies 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.

AutotrophsPhotoautotrophs- 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 bothObligate aerobes- constantly need oxygen Obligate anaerobes- live without oxgenFacultative anaerobes- live with/without oxgen EX: E. coli

Life CycleBacteria 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

Alexander Fleming and PenicillinAntibioticsAntibiotic resistance

Bacteria’s impact on life

Pathogenic bacteriaToxinsBiowarfareProduction of chemicals and foodsMining, environmental useNitrogen fixationDecomposers

What Are The Basic Tools Of Biotechnology?

Micropipets

Gel boxes

Micropipets Used for accurately measuring very

small amounts of fluids Used in almost all biotechnology to

measure 1 μL = .001 mL = .000001 L = 1 x

10^-6 L 2 mL = μ L = L

Pipet typesP-20 (2-20 μ L) P-200 (20-200 μ L) P-1000 (200-1000 μ L)

Reading quiz

Viruses contain a coat made up of ____ and their genetic material is _____

Lytic and lysogenic are examples of what?

How is HIV different than many other viruses?

The problem…

A disease is causing large pale spots on the leaves of tobacco plants

1892, Ivanovski identified the cause of the disease to be in the juice

1897, Beijerinck suggested that tiny particles caused the disease, and named them viruses (Latin-poison)

Viral structure

Life cycles

HIV and AIDS

HIV is the virus that can lead to AIDS Transmission Prevention

AVOID any of the forms of transmission!

HIV mutates, evolves very rapidly Evades defenses of the immune system Destroys Helper T cells

Is it living?

Contains genetic material? YEPEvolves? YEPInteracts with other organisms? YEP

(parasitic)Metabolic activity? IN A HOST CELLReproduces? IN A HOST CELLWhat do you think?

Reading quiz (get SG 3,4C)

Write the term that best represents each description 1. Circular piece of DNA that replicates

independently of the chromosome(s) 2. Protein that can “cut” DNA at precise spots 3. Purposeful manipulation of DNA 4. Overhanging sequence of bases left after DNA is

“cut” 5. Substance that can be used to identify cells with

our gene of interest

1 point each

1. Circular piece of DNA that replicates independently of the chromosome(s)

2. Protein that can “cut” DNA at precise spots

3. Purposeful manipulation of DNA

4. Overhanging sequence of bases left after DNA is “cut”

5. Substance that can be used to identify cells with our gene of interest

Plasmid (vector okay)

Restriction enzyme

Genetic engineering

Sticky end

Antibiotic (tetracycline okay)

Genetic engineering defined:

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

Or… as dramatic as DNA from different kingdoms!

Recombination of genetic information bytransferring genetic information from one organismto another, usually by way of a recombinant “vector”

AnimalPlantBacteria

Uses in basic research

Simple: - to produce multiple copies of DNA - to produce multiple copies of a

protein

More complex:

- monitor spread of virusinfecting tobacco plants

Transgenic organisms Organisms contain

genes from other organisms Microorganisms Animals Plants

Tools to accomplish genetic engineering

Restriction EnzymesProtein molecules used to cut DNA

Ligase EnzymesProtein molecules used to put together DNA

PlasmidsSmall DNA molecules used to put new genes

into bacteriaViruses

Used to put new genes into other living things

How is DNA cut?

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

Eco RI, Hind III Restriction enzymes cut DNA at specific four or six-

base-pair-sequences called restriction sites. Examples for different restriction sites:

Eco RI at G AATT C C TTAA G Hind III at A AGCT T T TCGA A

How is a gene inserted?

Gene therapy

1. Identify gene 2. Clone gene 3. Replace the

faulty or missing gene

4. Result? The correct protein should eliminate cause of the disorder Advantages Disadvantages

Transformation

Griffith experimentOutside DNA gets incorporated into

bacteriaWhat you have: pGLO plasmid (outside

DNA) and E. Coli (bacteria)

Transformation procedure

How do you get pGLO into E. Coli?

How do you grow transformed E. Coli?

How do you identify transformed cells?

Compentency CaCl2 Heat/ice shock

Spread E. Coli on petri dish with agar and incubate

Amp resistance Arabinose GFP protein

pGLO

Origin of plasmid replication

ON/OFF switch

Green Fluorescent Protein

Beta-lactamase to break down ampicillin

araC

GFP

bla

ori

A plasmid used as a vector must have

a. An origin of replication

b. A gene conferring resistance to an antibiotic.

In addition, pGLO contains

c. The Green Fluorescent Protein - the protein of interest

d. An ON/OFF switch that controls the synthesis of GFP by the presence or absence of the sugar, arabinose.

Tranformation prelab

Read thoroughly through WB 204-206 Make notes on any procedures you don’t

understand. Highlight important information Use the background information to answer the

following 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

LB + DNA : Prediction, why?

LB + DNA + amp : Prediction, why?

LB + DNA + amp + arabinose : Prediction, why?

Satellite colonies

Lab report

Introduction Purpose Deduction Background info

Materials/procedures Include amounts SUMMARIZE procedures (don’t copy from lab)

Results Data table, picture, bar graph?, written paragraph

Analysis 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”

As we’ve done before, take turns reading through the article (rotate after each paragraph). Each person will have a different role Summarizer: Summarizes EACH paragraph read Predictor: Predict what will happen next Connector: Discusses connections with class

content/experiences/society Questioner: Asks under and on the surface questions. Asks if

anyone else has questions

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)

Write the term the best represents each description1. A weakened or killed pathogen2. A disease when the body launches an

immune response against its own cells3. The first one was produced by Jenner

using cowpox4. The body system HIV attacks

Antigen vs. antibody

Vaccines

A weakened or killed virus is injected into the individual

Elicits an immune response (antibodies) Does not harm the individual

Gel Boxes

Used to separate chemicals like DNA based on size, charge, etc..

Used for identification/DNA fingerprinting, researching genes, etc..

Lab B analysis

Voltage vs. current When was electricity

being conducted? Why? Electricity allows

electrolysis to occur Role of phenol red? Role of buffer? Requirements for running

DNA through a gel: Current and a stable pH

H2O + NaCl + phenol red + TAE buffer

Note bubbles and swirls of changing pH

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

Charge of DNA: ____ If put into an electric field, it will move from the _____ to the

____ pole. A gel serves as the porous matrix for the movement of the DNA

molecule. DNA is cut into fragments of different size.

DNA GEL

RFLP (DNA FINGERPRINTING) Protocol

libpro

to

Polymerase Chain Reaction (PCR)

Purpose: Amplify DNA quickly

Applications: Acquire enough DNA to run a DNA fingerprint

Uses taq polymerase

Mammalian cloning Nucleus of an

organism’s egg is destroyed (lets say a sheep)

Nucleus is removed of a donor organism of the same species and put into the egg

That egg then develops into a clone of the donor’s DNA

Issues about cloning

Cloning already happens in nature (identical twins, bacteria, asexual reproduction)

What are consequences of mammalian cloning?

How easy is it to do? Cloning the sheep Dolly took 277 attempts. What happens to unsuccessful eggs?

Currently, there is a ban on human cloning