Agrobacterium tumefaciens - Connecticut's BioBus

jamaicanabsorbingBiotechnology

Dec 5, 2012 (4 years and 6 months ago)

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© 2010 BioScience Explorations

Welcome to our professional development
workshop

© 2010 Bioscience Explorations

What is CURE?

Connecticut United for Research Excellence, Inc.



Represent biotechnology and pharmaceutical sectors before state
legislature and policy makers



Build a critical mass of biotech and pharmaceutical companies



Foster relationships between academic and industry research that lead
to technology transfer



Be the “go to” source for information about bioscience in
Connecticut


© 2010 Bioscience Explorations

In Connecticut

Achillion Pharmaceuticals, Inc.

Alexion Pharmaceuticals, Inc.

Boehringer Ingelheim Pharmaceuticals

Bristol
-
Myers Squibb

CGI Pharmaceuticals

CuraGen Corporation

HistoRx, Inc.

Institute for Pharmaceutical Discovery

Invitrogen Corporation

Marinus Pharmaceuticals, Inc.

Molecular Staging

Neurogen Corporation

Pfizer

Purdue Pharma L.P.

Rib
-
X Pharmaceuticals, Inc.

© 2010 Bioscience Explorations

Bioscience Careers


Connecticut Business and Industry Association

http://www.cbia.com/ed/STC/career_explorations/career_explor_info/health_b
io.htm


“Biotechnology and pharmaceutical
companies are among the fastest
-
growing industries in Connecticut,
yet they are having difficulty finding
qualified workers with the right
technological and scientific skills to
fill high
-
demand, high
-
wage jobs.”
-

CBIA


© 2010 Bioscience Explorations

Sponsors and Contributors

CURE and Connecticut Innovations

Pfizer Inc and The Pfizer Foundation

Boehringer Ingelheim

Achillion Pharmaceuticals,

State of Connecticut

Wesleyan University

Quinnipiac University

University of New Haven

Manchester Community College

Yale University


SMART Technologies

Bio
-
Rad

Invitrogen

Edvotek

Fisher Scientific

Antarus Biotech Inc.


© 2010 Bioscience Explorations


Generate student interest in and excitement for
bioscience to encourage career exploration



Provide educators with laboratory experience, bioscience
information and innovative teaching techniques



Generate public understanding, enthusiasm and support
for bioscience


CURE’s Educational Goals:

© 2010 Bioscience Explorations

Spring 2010

© 2010 Bioscience Explorations

Since 2004:



Trained over 150
teachers



Reached over 27,000
students




BioConnection Accomplishments

© 2010 Bioscience Explorations

Today We Will:


Provide information for a BioConnection visit




Reinforce and increase your knowledge of
bioscience




Prepare you to use BioConnection PCR in your
curriculum


© 2010 Bioscience Explorations

Genetically Modified Organisms
(GMOs)


In this experiment, students will
test soy to determine if it has
been genetically modified with
a drought resistance gene.
Students will use a scientific
technique called polymerase
chain reaction (PCR) to amplify
a specific portion of the DNA to
determine whether their product
is in fact a GMO.

© 2010 Bioscience Explorations

Setting up and Using the Module


Refer to your module guides


Read thoroughly before using module



2 modules: Edvotek and Bio
-
Rad


The same except for Thermal cyclers and
micropipettes


© 2010 Bioscience Explorations

DNA Extraction

1.
Using your plastic transfer pipette, transfer all of your
saline
solution

to the tube containing the
soy flour
.


2.
Using your micropestle,
grind the flour and water mixture

well for
one full minute
. Grind the pestle against the bottom
of the tube to help break down the flour (clumps on the
bottom are ok).
Teachers should now pass out one tube of
Chelex per student.


3. Using your sharpie, label the 1.5 ml tube containing the
Chelex

clearly with your
initials

on the top of the tube.

© 2010 Bioscience Explorations

DNA Extraction

4. Using the same plastic transfer pipette, transfer about 5
drops of the flour/water mixture to the labeled tube
containing the Chelex.
Cap the tube tightly
. Flick the tube
a few times to mix the contents.


5. Carefully place your labeled tube, containing your food
sample/Chelex mixture, into the heat block at 56
o
C for five
minutes.


6. After five minutes, carefully remove your tube from the heat
block. Flick the tube a few times to mix the contents.
Centrifuge your tubes for one minute
.

© 2010 Bioscience Explorations

Set up your PCR Tube

7. Using your micropipette, transfer 10
m
l of your DNA
supernatant (the liquid on top) directly into your tube labeled
H
2
0. Close the tube and flick gently to mix.
This is your
diluted DNA template.



8. Using a sharpie, label the
top
and

side

of a small PCR tube
clearly with your initials. Be careful not to disturb the
contents in the bottom of the tube.


9. Using your micropipette, transfer
10
m
l

of your diluted DNA
sample into the small PCR tube. Be careful not to disturb the
contents in the bottom of the PCR tube.


10. Close your PCR tube securely. Place your tube inside the
thermal cycler.

© 2010 Bioscience Explorations

Logistics

© 2010 Bioscience Explorations



Make sure all paperwork is in, especially principal letter



Module is dropped off to your school’s
main office



We’ll give you a one hour delivery window


Someone must sign for equipment, place samples in freezer


Do not plan to use equipment on drop off or pick up days



You will have the module for approximately 2 weeks



Staff is usually available to
help

you run your first experiments
if needed; but please give us as much notice as you can






Before BioConnection

© 2010 Bioscience Explorations


You can request samples for up to 98 students working in
pairs

(i.e. 48 sets of samples)



Please request only what you can reasonably use


Extra samples can sometimes be accommodated at cost (supplies fee)



24 students can work at a time (12 stations)



Experiment can be conducted over 2
-
3 class sessions



Potential stopping points are noted in the module guide


Have someone to help you run these labs if possible!


Before BioConnection

© 2010 Bioscience Explorations

After BioConnection



When returning, please
fill out and sign checklist



Please pack modules correctly, according to pictures on bin lids



Contact us
before

pick
-
up day if anything breaks

or is
missing (modules are often restocked on the road)



Have module ready to go in the school’s
main office

for pick
-
up


One hour pick
-
up window



© 2010 Bioscience Explorations

After BioConnection



Sign up again on our website (you will
not

automatically be
slated in every year unless you keep signing up)



Address any thank you notes from students etc. to
“BioConnection Sponsor” and send to our New Haven office



Training must be renewed every two years (modules and
curriculum change somewhat every year)

© 2010 Bioscience Explorations

Safety


Check for soy allergies before conducting this lab



Review teacher and module guides for full safety info



Safety goggles are required


We have a limited number of sets available to borrow but you must specifically
request them beforehand



Gloves (non
-
latex) and aprons are recommended



Students may not take any materials home



A trained teacher must be present at all times that the equipment is in use


© 2010 Bioscience Explorations

SMART Notebook Files



Access Notebook files at
www.bioscienceexplorations.org



Slides go along with the experiment



Download Notebook Interactive viewer from
SMART (link is available on our website)


© 2010 Bioscience Explorations

Genetically Modified Organisms


Organisms that are modified by the
alteration or introduction of
foreign DNA, in order to produce a
desired trait or effect



Plants are the most common
targets (transgenic crops):


Pest resistance (e.g. Bt
-
hybrid corn)


Drought resistance


Cold
-
tolerance


Shelf
-
life


Ripening


Nutritional value




Bt
-
hybrid vs. non
-
Bt
-
hybrid corn

© 2010 Bioscience Explorations

Creation of a GMO


Genetic material is artificially combined,
usually in the form of circular pieces of
DNA called
plasmids


Plasmids contain not only DNA coding for
the trait of interest, but also DNA that
controls the expression and selection of the
trait



Delivery methods:


Agrobacterium tumefaciens


Ballistics or gene gun


Electroporation


Chemical and/or heat treatment




http://sustain.no/virtue/newsletter/00_08/curr
-
trant/more
-
info/plasmid.jpg

© 2010 Bioscience Explorations

Restriction enzymes



Isolated from bacteria




Microscopic scalpels




Cut DNA molecules at
specific sequences known as
“recognition sites”

…G

Eco R1: from
E. coli
,
strain R1


Cuts the

Following:


…G A A T T C…

…C T T A A G…

A A T T C…

….C T T A A

G…

© 2010 Bioscience Explorations


Process by which genetic material carried by an individual cell
is altered by the addition and expression of foreign DNA



The DNA may or may not be incorporated into the genome



Electrical, thermal, mechanical and chemical and
biological

methods




Transformed cells are identified by some type of marker or trait


Antibiotic resistance


Color

Transformation

© 2010 Bioscience Explorations

Transformation using
Agrobacterium tumefaciens



Bacterial species




Normally infects plants and
causes crown
-
gall disease




Very efficient at inserting DNA
into plant cell genome


Bingo!


© 2010 Bioscience Explorations


T
-
DNA leads to synthesis of:


Auxin


plant hormone causes cell proliferation


Opines


serve as a source of energy for
A. tumefaciens

Symbiotic Relationship

© 2010 Bioscience Explorations

Step 1. Engineer
Agrobacterium

with
your favorite gene(s)….

Ti = Tumor inducing

Step 2. Disarm the genes that cause crown
gall disease

© 2010 Bioscience Explorations

http://dragon.zoo.utoronto.ca/~jlm
-
gmf/T0301C/images/transgenicflowchart.jpg

Step 3: Infect plant cells with engineered
Agrobacterium…

© 2010 Bioscience Explorations

Recombinant DNA Animation

© 2010 Bioscience Explorations

PBS Harvest of Fear

© 2010 Bioscience Explorations

Detecting a GMO


ELISA
-

Uses antibodies to detect
the presence of foreign proteins



Processing can sometimes destroy the
protein



PCR

-

detects and amplifies foreign
DNA



More sensitive than ELISA


Can be used on processed foods


More general: usually detects common
plasmid element such as 35S
promoter



Photo: John Morris

GMO

Promoter
: a regulatory region of DNA located upstream (towards the 5’ region) of a gene, providing a
control point for regulated gene expression.

© 2010 Bioscience Explorations

PCR


P
olymerase
C
hain
R
eaction


Goal: Amplify a
target

sequence of
DNA



Mimics the natural process of DNA
replication



Replicates a DNA segment in
minutes by repeated heating and
cooling of reaction tubes

© 2010 Bioscience Explorations

PCR Components


DNA template (starting material)



Two Primers (segments of DNA that define the region to be
copied)



Taq polymerase (from the bacterium
Thermus aquaticus)



Free nucleotides (dNTPs) A T G C



Buffer containing Mg
2+



Thermal cycler



© 2010 Bioscience Explorations

DNALC PCR Animation

© 2010 Bioscience Explorations

Bio
-
Rad PCR Animation

© 2010 Bioscience Explorations

DNAi

© 2010 Bioscience Explorations

© 2010 Bioscience Explorations

Gel Electrophoresis


What does it mean?


Gel

-

scientific Jell
-
O

Electro

-

electricity

Phoresis



to carry across

Gel electrophoresis is using electricity to carry molecules
across a gel


© 2010 Bioscience Explorations

Agarose Gel

50,000x magnification.

© 2010 Bioscience Explorations

The Process


Samples are loaded into
wells


Electric current is passed
through gel


Gel acts as a maze for
molecules


Molecules are separated




How?

-

+

© 2010 Bioscience Explorations

Gel Electrophoresis Virtual Lab

© 2010 Bioscience Explorations

Pre
-
lab Activities


PCR Introduction


Learn.genetics PCR website


Paper clip PCR activity



DNA fruit extraction



on our website



Bioinformatics: Electronic PCR


on our website




© 2010 Bioscience Explorations

PCR Virtual Lab

© 2010 Bioscience Explorations

Paper Clip PCR

© 2010 Bioscience Explorations

Post
-
lab Activities



GMOs in Medicine: GMOs for Diabetics?


webquest on DNAi.org website




CAPT Generation III Embedded Task




Class debate with focus statements




Bioethics discussions


© 2010 Bioscience Explorations

DNAi.org


Insulin Production

© 2010 Bioscience Explorations

Debate Focus Statements


Labels should be placed on all products containing
genetically modified plant products.



Genetically modified foods have made our lives
easier and better.



Genetically modified plants are tastier and healthier
than non
-
modified plants.



Genetically modified foods pose no threat to the
environment.


© 2010 BioScience Explorations

Run PCR Products

© 2010 Bioscience Explorations

Double Helix Game

© 2010 Bioscience Explorations

Online Transcription/Translation
Activity

© 2010 Bioscience Explorations

BLAST

© 2010 Bioscience Explorations

35S Promoter Sequence

Forward

Primer

Reverse

Primer

gctcctacaa

atgccatca
t tgcgataaag gaaaggccat cgttgaagat gcctctgccg
acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc
caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta aggga
tgacg

cacaatccca ctatc

Amplicon: 195 base pairs

© 2010 BioScience Explorations

Questions?

Thank you for attending our PCR workshop!