Biotechnology

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14 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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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
diseases


Genetically 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 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

CO
2
.

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


Alexander Fleming and Penicillin


Antibiotics


Antibiotic resistance

Bacteria’s impact on life


Pathogenic bacteria


Toxins


Biowarfare


Production of chemicals and foods


Mining, environmental use


Nitrogen fixation


Decomposers

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
μ
L = .001 mL = .000001 L = 1 x
10^
-
6 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


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? YEP


Evolves? YEP


Interacts with other organisms? YEP
(parasitic)


Metabolic activity? IN A HOST CELL


Reproduces? IN A HOST CELL


What 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 by

transferring genetic information from one
organism

to another, usually by way of a recombinant
“vector”

Animal

Plant

Bacteria

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


Organisms contain
genes from other
organisms


Microorganisms


Animals


Plants

Tools to accomplish genetic engineering


Restriction Enzymes


Protein molecules used to cut DNA


Ligase Enzymes


Protein molecules used to put together DNA


Plasmids


Small DNA molecules used to put new
genes into bacteria


Viruses


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 experiment


Outside DNA gets incorporated into
bacteria


What 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

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
description


1. A weakened or killed pathogen


2. A disease when the body launches an
immune response against its own cells


3. The first one was produced by Jenner
using cowpox


4. 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

H
2
O + 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

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