Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
Lecture 6
Some More Techniques in Biotechnology
© life_edu
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
Kimberly Nelson
OnCampus Live
BCH 190, MIC 190, AFS 190, NRS 190, PLS 190
OnLine BCH 190
A Sweeping General Survey on Life and Biotechnology
A Public Access College Course
The University of Rhode Island
Issues in Biotechnology:
Biotechnology, Our Society and Our Future
life
edu.us
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
Kimberly Nelson
BCH 190
Section I. The Mechanics of Life and General
Biotechnology
Section II. The Applications of Biotechnology
A Sweeping General Survey on Life and Biotechnology
A Public Access College Course
© life_edu
The University
of Rhode Island
life
edu.us
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
5. Trends, Patterns and Relationships in Biology
6. Some More Techniques in Biotechnology
A Sweeping General Survey on Life and Biotechnology
A Public Access College Course
© life_edu
The University
of Rhode Island
Issues in Biotechnology:
The Way We Work With Life
Dr. Albert P. Kausch
life edu.us
The Mechanics of DNA
Lecture 6
Some More Techniques in Biotechnology
© life_edu
Are there any foods or ingredients that
you have avoided or eaten less of?
(A)
(B)
(C)
(D)
yes
no
do know
refuse to answer
50
40
30
20
© life_edu
10
0
1
2
3
4
Half of consumers are avoiding some
food/ingredient…
Are there any foods or ingredients that you have avoided or eaten less of?
100%
80%
60%
Don't know / refused
No
Yes
40%
20%
A
pr
-0
3
Ja
n04
M
ar
-0
5
2
ug
-0
A
-0
1
Se
p
Ja
n-
01
0%
© life_edu
If yes, what foods or ingredients did you
avoid or eat less of?
Foods/ingredients avoided
(A)
(B)
(C)
(D)
(E)
sugar / carbohydrates
fats / cholesterol
animal products
salt / spices
biotechnology products
30
25
20
15
10
© life_edu
5
0
1
2
3
4
5
…it’s not biotech!
If yes, what foods or ingredients did you avoid or eat less of?
(Open-ended; Multiple responses allowed, n = 478)
Foods/ingredients avoided
3/05
Sugar/Carbohydrates
58%
Fats/Cholesterol
37%
Animal Products
34%
Salt/Spices
14%
Snack Foods
11%
Biotechnology
<½%
© life_edu
IFIC 2005
FDA requires special labeling when a food is produced
under certain conditions: when biotechnology’s use
introduces an allergen or when it substantially changes the
food’s nutritional content... Otherwise special labeling is
not required. Would you say that you support or oppose
this policy of FDA?
(A)
(B)
(C)
(D)
(E)
support
oppose
neither support or oppose
don’t know
refuse to answer
40
30
20
© life_edu
10
0
1
2
3
4
5
Current FDA labeling policy
supported by majority
FDA requires special labeling when a food is produced under certain conditions: when
biotechnology’s use introduces an allergen or when it substantially changes the food’s
nutritional content... Otherwise special labeling is not required. Would you say that you
support or oppose this policy of FDA?
80%
70%
60%
Don't
know/refused
50%
Neither support
nor oppose
40%
Total oppose
30%
20%
10%
19
9
Fe 7
b9
O 9
ct
M 99
ay
-0
Ja 0
n0
Se 1
p0
A 1
ug
-0
A 2
pr
-0
Ja 3
n0
M 4
ar
-0
5
0%
Total support
It is now possible to clone
any gene from any
organism and move it
into plants
© life_edu
Plasmids are circular pieces
of DNA found in some bacteria
Many copies per cell
Antibiotic resistance gene
Plasmids can be cut and
pasted back together
Foreign genes can be
inserted
© life_edu
How is a gene cloned?
Boyer, Cohen, and Berg, 1972
Enzymes were discovered
that cut DNA
at specific sequences
And subsequently,
enzymes were discovered
that paste DNA together
The ability to cut and
paste DNA
allowed gene cloning
How is a gene cloned?
Foreign DNA (gene)
is inserted into a plasmid
that has a gene for
antibiotic resistance
The plasmid is introduced into
a bacterial cell and grown on
the antibiotic
Only bacteria with the plasmid
grow…the inserted gene is
copied many times
Anatomy of a Transgene
Promoter
Coding Sequence
Protein coding sequence
Cell specificity
Developmental specificity
Start transcription
Terminator
Stop transcription
Message stability
Gene constructs can be moved into plants and the gene is expressed
driven by the promoter sequence
© life_edu
Agricultural Biotechnology
Genetically Modified Foods:
Panacea
Or Pandora’s Box?
© life_edu
Agricultural Biotechnology
Genetically Modified Organisms GMO
How is it done?
It is now possible to
clone any gene
from any organism
and move it into
plants
© life_edu
A Success
Story:
Genetically
Modified
Salmon
Constitutive expression of
rainbow trout growth factor
hormone
All salmon shown are fourteen
months old, those at the bottom
are the controls


Transgenic Atlantic
salmon produced by
Aqua Bounty Inc.
Growth rate, not ultimate
size is enhanced.
Commercial production
of Aqua Bounty salmon is
being reviewed by FDA.
First transgenic meat
product.
Would you order genetically modified salmon at a
restaurant if you also had a choice of wild salmon?
(A)
(B)
(C)
(D)
yes
no
doesn’t matter
undecided
40
30
20
10
0
1
2
3
4
Tools and Techniques
used in Biotechnology
Gene transfer from one organism to another is not new
Image of two species of
bacteria transferring viral
phage particles
Bacteria transfer genes
to other bacteria and plants
Now in nature there
is another organism
capable of
Transferring DNA:
we call that organism
a human being
Gel Electrophoresis:
the separation of molecules,
DNA, RNA and proteins
by charge and size
Electro refers to the energy of
electricity. Phoresis, from the
Greek verb phoros, means
“to carry across.” Thus, gel
electrophoresis refers to the
technique in which molecules
are forced across a span of gel,
motivated by an electrical
current.
Applications of Gel Electrophoresis
• DNA Fingerprinting
• DNA Recombinant Technology
• Forensics
• The Human Genome Project
DNA carries a net negative charge; it
is negatively charged because the
phosphates (red circles) that form the
sugar-phosphate backbone of a DNA
molecule have a negative charge.
As the separation process continues, the
separation between the larger and smaller
fragments increases.
• Molecular weight markers are often electrophoresed
with DNA.
• Molecular weight markers are usually a mixture of
DNAs with known molecular weights.
• Molecular weight markers are used to estimate the
sizes of DNA fragments in a DNA sample.
The Techniques of
Molecular Biotechnology
Technology has created new Fields
DNA detection
DNA synthesis
DNA sequencing
DNA cloning
Genomics
Bioinformatics
Pharmacogenomics
Transgenics
Expression cassette
construction
Computational
Biology
RNA detection
Population Genetics
Protein detection
Proteomics
Techniques of
Molecular Biotechnology
• Polymerase Chain Reaction
• Southern Blot Analysis
• Northern Blot Analysis
• Western Blot Analysis
• cDNA Library Construction
• DNA Sequencing
• Gene Isolation
• Gene Vector Construction
PCR
PCR
The Polymerase Chain Reaction
PCR
The Polymerase Chain Reaction
You Leave a Piece of You Behind
PCR was used on the “BLUE DRESS”
and showed President Clinton's association with
Monica Lewinsky.
Polymerase
Chain Reaction
Nobel laureate in Chemistry,
1994
PCR is Amplification
 THERE ARE MILLIONS OF DIFFERENT GENES OR
SEQUENCES WITHIN ANY DNA SAMPLE (BLOOD,
TISSUE, PLANT, ETC.).
 A SPECIFIC SEQUENCE IS SELECTED TO BE
AMPLIFIED (RED ABOVE). THIS SEQUENCE CAN BE ANY
GENE OF INTEREST OR A NON-CODING MARKER
REGION OF DNA.
IN ORDER TO COPY THE SEQUENCE OR GENE, A
SHORT SEQUENCE ON EITHER SIDE OF THE
SECTION MUST BE KNOWN. THIS REGION (BLUE
ABOVE) WILL SERVE AS A PRIMER ATTACHMENT
SITE TO COPY THE DNA TARGET SEGMENT.
IN ORDER TO AMPLIFY A SPECIFIC FRAGMENT OF DNA,
SEVERAL THINGS ARE NEEDED, INCLUDING PRIMERS
AND DNA POLYMERASE.
AN ENZYME WHICH COPIES DNA, PRIMERS ARE SHORT
PIECES OF DNA OR RNA DESIGNED TO PAIR WITH
GENOMIC DNA AT A SPECIFIC ATTACHMENT SITE FOR
THE MAIN PURPOSE OF HELPING THE DNA
POLYMERASE BIND AT THE DESIRED SECTION.
NUCLEOSIDE TRIPHOSPHATES, THE BUILDING BLOCKS
OF DNA ARE ALSO NEEDED.
EACH NUCLEOSIDE TRIPHOSPHATE CONSISTS OF:
 A BASE (ADENINE, THYMINE, CYTOSINE
OR GUANINE).
 A SUGAR AND THREE PHOSPHATES.
WITHOUT A SHORT PIECE OF DNA(OR
RNA) TO ATTACH TO, DNA POLYMERASE
CAN NOT COPY A DNA STRAND.
PCR REQUIRES SEVERAL CYCLES OF AMPLIFICATION. EACH CYCLE CONSISTS OF
THREE TEMPERATURE CHANGES.
 THE STARTING TEMPERATURE (95 C) SEPARATES THE DNA STRANDS.
 A LOWERED TEMPERATURE (50-60 C) ALLOWS PRIMERS TO BIND TO
COMPLEMENTARY SEQUENCES IN THE DNA.
 A SLIGHTLY HIGHER TEMPERATURE (72 C) ALLOWS DNA
POLYMERASE TO ATTACH TO THE PRIMERS AND COPY THE DNA
STRANDS (EXTENSION).
DNA STRANDS ARE SEPARATED BY HEATING @ 94o C.
THE TEMPERATURE IS LOWERED TO 54oC TO
ALLOW PRIMERS TO PAIR WITH
COMPLEMENTARY DNA SEQUENCES.
MAKING NEW DNA MOLECULES:
 DNA POLYMERASE ATTACHES TO THE PRIMERS @ 72 C.
 DNA POLYMERASE ADDS NUCLEOSIDE TRIPHOSPHATES
TO THE PRIMERS TO COPY THE DNA STRANDS.
COPYING IS COMPLETED FOR EACH STRAND.
THE PROCESS IS REPEATED IN THE NEXT CYCLE.
THE TEMPERATURE IS RAISED AGAIN TO SEPARATE
THE DNA STRANDS.
THE TEMPERATURE IS LOWERED TO ALLOW PRIMERS
TO ANNEAL. DNA POLYMERASE ATTACHES TO
THE PRIMERS AND DNA IS COPIED TO MAKE
4 STRANDS OF DNA.
The stability of TAQ Polymerase allows for this
amplification process through the three temperature
changes.
THE PROCESS OF COPYING DNA STRANDS IS REPEATED
32-35 TIMES. WITH EACH AMPLIFICATION CYCLE,
THE NUMBER OF COPIES OF THE DNA SEQUENCE IS
DOUBLED UNTIL MILLIONS OF COPIES HAVE BEEN
MADE.
Issues in Biotechnology
A method used to copy small amounts of DNA many times over
was invented by Dr. Kary Mullis in the 1980s and is called
PCR. PCR stands for:
(A)
(B)
(C)
(D)
(E)
Protein Chromosomal Replication
Polymerase Chain Reaction
Pipetman California Reaction
Peptide Catalytic Reactors
Polysaccharide Catalyst Repair
70
60
50
40
30
20
10
0
1
2
3
4
5
Forensic applications of
DNA based technologies
•
Fingerprinting
OJ Simpson
•
Identification
•
Paternity
•
Crime Solving
•
World wide
data base
Forensic Identification:
Basic Principles
Each of us is genetically unique
If enough genetic variation is tested, each
of us can be uniquely identified
DNA is found in nearly all cells (blood,
semen, hair, etc.)
DNA from an evidentiary sample can be
matched with DNA from a suspect to
implicate or exonerate
STRs Are Used in Identity Testing
“Short
Tandem
Repeat
sequence”
...atatatacaacttactaccatata
ccgattacgatcgaattataccgcgga
cgtagtaatgacgatgaagtaactata
tatatatatatatatatatatatatat
atatatatatatatatatatatatata
tatatatatatatatatatatatatat
atatatatatatatatatatatatata
tatatatatatatatatatatatatat
atatatatatatatatatatatatata
tatatatatatatatatatatatatat
atactacctaccagggaggagata...
Laboratory PCR Instrument
INPUT:
Sample DNA, PCR enzymes,
primers, individual nucleotide
building blocks (and maybe
fluorescent labels)
OUTPUT:
Specific DNA fragments amplified
millions of times for easy visualization
With sizes that vary between individuals
After PCR, DNA Fragments are
Separated on a Gel
PCR products for each sample
DNA fragments move through
gel based on their size
-
TIME
45
180
40
170
35
160
30
150
25
140
20
130
15
120
10
110
5
+
Minutes
100
Fragment
Size
Identity Testing Using PCR
Analysis of four different sections of the DNA
Possible conclusions:
S = size standards
V = victim’s DNA
1 = suspect #1 blood
2 = suspect #2 blood
3 = suspect #3 blood
E = evidence #1
S = size standards
A. Suspect 1 DNA was
at the scene
B. Suspect 2 DNA was
at the scene
C. Suspect 3 DNA was
at the scene
D. None were at the scene
E. Multiple suspects
were at the scene
F. Data is inconclusive
S V 1
S = size standards
V = victim’s DNA
1 = suspect #1 blood
2 = suspect #2 blood
3 = suspect #3 blood
E = evidence #1
S = size standards
2 3
E S
450
400
350
300
250
200
150
100
50
A complete match!
PCR was used on the “BLUE DRESS”
and showed President Clinton's association with
Monica Lewinsky.
Issues in Biotechnology
DNA tests exclude Martin from gun
grip in Zimmerman case September 19, 2012
Another round of evidence was released Wednesday in the
second-degree murder case against George Zimmerman,
including forensic tests that show Zimmerman’s DNA was
the only one that could be identified on the grip of the gun
used to shoot 17-year-old Trayvon Martin.
Zimmerman’s DNA also was identified on the gun’s holster.
The tests were inconclusive as to whether Martin’s DNA was
on the gun’s holster.
Congratulations!!!!!
$100,000.00!!!!!!!
You have
To Invest in Biotech.
Issues in Biotechnology
Stock Project The idea is to select five biotechnology
companies and invest $100,000 (fictitiously, of course)
Issues in Biotechnology
Stock Project
You can spread your money evenly across five companies (i.e.
$20,000 each) or not. For example, if a company is trading at
$20/share you can purchase 1,000 shares for $20, 000. Look up
companies on Google (e.g.: type Monsanto stock) and record their
stock ticker designation (e.g.. MON of the New York Stock
Exchange, NYSE). Observe their performance over the past year.
Record current trading price per share and calculate how many
shares you can buy. You must choose your companies and the
number of shares. Submit a single page summary on these results.
Toward the end of the course you will look up these same
companies and determine the cost per share at that time. Calculate
your losses or gains for each company and your total losses and
gains. This project will be summarized at the end of the course in
a one page summary of losses and gains.
Biotechnology Stocks Project
Congratulations!!!!!
$100,000.00!!!!!!!
You have
To Invest in Biotech.
1. Select and Research five Biotech companies.
2. Print and submit the current stock quote and for each company.
3. Invest chosen amounts in each. Calculate the number of shares
in each (Submit a One page summary).
4. Contact company to receive investors information (Optional).
5. Monitor Stock during the course.
6. Calculate gains and losses. Submit report with final exam.
Biotechnology and
Industry
“In retrospect, recombinant-DNA may rank as the safest
revolutionary technology ever developed.”
- James D. Watson, Nobel Prize Winner, 1953
The Development of
Biotechnology is directly
linked with Industry
The application of the
biological sciences has moved
from academia to the private
sector
Driven by profits and the
promise of profits
The distinction between
Basic and Applied Science
is blurred
Basic science is immediately
applied in today’s biotech
Biotech Industry
Red vs Green companies
pharmaceuticals vs
agriculture
(blood vs chlorophyll)
Information Sciences
Genomics
Proteomics
Phenomics
Bioinformatics
Population Genetics
Clinical trials
The Role of Companies
in the Development of
Biotechnology
Technology Development
Patents
Markets and Products
Market-driven Innovation
Genomics
Companies
Celera
Incyte
Genome Therapeutics Corp
Millenium
Paradigm Genetics
DuPont
Genaissance
Monsanto
Technology
Companies
Invitrogen
Stratagene
Qiagen
Packard
PE Biosystems
Kairos
BioRobotics
Biotechnology
Industry
Advertisement of services
in international journals
NATURE
Science
Nature Biotechnology
“Picks and shovels
for the Gold Rush”
Innovative technologies
become biotech products
Big money in licenses
Commercialization of
biotech products
requires
Freedom to
Operate (FTO)
with all the
technologies
used
And look at the ads!
BioBelt
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Monsanto is an agricultural company. We apply innovation and technology to
help farmers around the world be successful, produce healthier foods, better
animal feeds and more fiber, while also reducing agriculture's impact on our
environment.
» View Biotechnology Videos
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Recent News
Mon, 17 Sep 2007 — Monsanto Increases Ongoing Earnings Per Share Guidance, Provides Free Cash Flow Guidance For Fiscal
Year 2007
Fri, 14 Sep 2007 — Monsanto, Dow Agreement Paves the Way for Industry’s First-Ever, Eight-Gene Stacked Offering in Corn
Thu, 13 Sep 2007 — Monsanto to Provide Information to Iowa Attorney General Concerning Seed, Trait and Chemistry Business
Agricultural Biotechnology
Stock Price
Stock Chart
| Annual Report
Stock Price
Monsanto
89.79
(MON)
Dow Jones
13,451.41
(DJIA)
-0.66
-6.14Stock Chart |
Annual Report
Pharmaceutical Biotechnology
NYSE: PFE 0.09
24.93
Stock Quote at: Sep 26 2012 12:22PM
Change
0.09
Vol
12,802,922
Day High
24.99
52 Wk. High 25.15
Day Low
24.79
52 Wk. Low 17.05
Open
24.91
Mkt. Cap(Bil)186.21
Prev Close 24.84
Stock Symbol: PFE Stock Exchange: NYSE
Stock Price
Stock Chart
| Annual Report
Biotechnology Stock Project
Example
Report 1
Henrietta Lack Student’s Name
PLS 190 Issues in Biotechnology
Professor Albert Kausch
Due Date 10-03-2012
Biotechnology Company
1. Intel Corporation (INTC) $22.05 per share / 900 shares = $19,845
2. Amgen (AGN)$55.66 per share / 350 shares = $19,481
3. Pfizer (PFE) $14.78 per share / 1,500 shares = $22,170
4. Apple Inc. (AAPL) $413.81 per share / $18,621
5. General Electric (GE) $15.38 per share / 1,292 shares = $19,870.96
Total = $99,987.96
Biotechnology Stock Project
Example
Report 1
Student’s Name Jack Straw
BCH 190 Issues in Biotechnology
Professor Albert Kausch
Date Due October 3, 2012
Biotechnology Company
Amgem Inc. AMGN: 56.04 per share 356 shares for $20,000
Pfizer Inc PFE: 17.84 per shares 1121 shares for $20,000
Monsanto Co MON: 65.86 per share 303 shares for $20,000
Bio Rad Laboratories Inc BIO: 89.50 per share 223 shares for $20,000
Syngenta AG SVJ: 205.30 Euros per share 71 shares for 14567.49
Euro or $20,000
Total; $100,000
Biotechnology Companies:
AstraZeneca
Active Motif
Aventis
Adventa
Celera PE
Incyte
Invitrogen
Pfizer
Merck
Smith Kline Beecham
Novartis
Monsanto
Qiagen
Roche
Paradigm Genetics
Chemicon International
Trevigen
Avigen
Metabasis Therapeutics
Pintex Pharmaceuticals
BioGen
Garst
Pioneer
DuPont
Pharmacia
Bristol-Myers
Squibb
Eli Lilly
Cistem Molecular Corp.
Operon Technologies
Biotechnology Companies:
DNX Transgenic Sciences
Chemdex
MWGAG BIOTECH
Double Twist
Larova Biochemie
Greiner Labortechnik
Sungene
Molecular Devices Corp.
Evotec BioSystems AG
BIACORE
TIB MOLBIOL
PE Biosystems
Bruker Daltonics Inc.
Eppendorf Scientific
Curagen
Cargill
Dow Agri Sciences
Dow Chemical Co.
AmGen
Bayer
Dynal
Noxxon Pharma
Schering
Boehringer
Rhein Biotech
Hyseq
Genome Therapeutics
O sweet spontaneous
earth how often have
the doting fingers of
purient philosophers pinched
and poked thee,
has the naughty thumb
of science prodded
thy beauty how
often have religions taken
thee upon their scraggy knees
squeezing and
buffeting thee that thou mightest conceive
gods
(but
true to the incomparable
couch of death thy
rhythmic lover
thou answerest
them only with
spring)
e.e. cummings
20. Of the following techniques, which would be most
unlikely to be used in a biotechnology laboratory?
(A)
(B)
(C)
(D)
(E)
gel electrophoresis
PCR
DNA cloning
use of the Hubble telescope
antibiotic resistant bacteria
21. Many of the molecular reactions used in
biotechnology occur in volumes less than a milliliter.
A Pipetman is:
(A) the new biomedical device made by tissue engineering and
now used to treat the damaged blood vessels of heart attack
victims
(B) a radical group of bioengineered superheroes in the
Hollywood movie GATTACCA
(C) a molecular biology tool used in the lab to measure small
volumes of liquid
(D) a new type of bio-engineered crop plants that are drought
tolerant
(E) a new surgical tool used in to extract cancer cells
22. Gel Electrophoresis is used for:
(A) the separation of molecules, DNA, RNA and proteins by
charge and size
(B) separation of various cell types in blood samples
(C) viewing cells at a high magnification
(D) as a home pregnancy test
(E) fusing cells during the process for cloning animals
23. Every time a cell divides it copies all of its DNA.
A method used commonly in many applications of
biotechnology today is called PCR. PCR:
(A) is used to study life on other planets
(B) stands for the PolyChromal Repercussions that occur in cell
division
(C) is a type of digital processing used in DNA sequencing
(D) uses a heat stable DNA polymerase to copy DNA
(E) is a dangerous prescription drug
24. Basic Forensic principles include:
(A) each of us is genetically unique
(B) if enough genetic variation is tested, each of us can be
uniquely identified
(C) DNA is found in nearly all cells (blood, semen, hair, etc.)
(D) DNA from an evidentiary sample can be matched with DNA
from a suspect to implicate or exonerate
(E) all of the above
25. Given DNA samples from three suspects, the
victims DNA and DNA evidence from a crime scene
the possible conclusions are:
(A) suspect 1’s DNA was at the scene; or suspect 2’s DNA was at
the scene; or suspect 3’s DNA was at the scene
(B) none were at the scene
(C) multiple suspects were at the scene
(D) data are inconclusive
(E) any of the above
26. A method used to copy small amounts of DNA
many times over was invented by Dr. Kary Mullis in
the 1980s and is called PCR. PCR stands for:
(A)
(B)
(C)
(D)
(E)
Protein Chromosomal Replication
Polymerase Chain Reaction
Pipetman California Reaction
Peptide Catalytic Reactors
Polysaccharide Catalyst Repair
27. STR stand for:
(A)
(B)
(C)
(D)
(E)
Separation of Trans Replicators
Scientists for True Religion
Short Tandem Repeats
Standard Test for Recidivism
Seek To Reach assay
28. The development of Biotechnology is directly
linked with Industry. Which of the following is not
true?
(A) the application of the biological sciences has largely moved
from academia to the private sector
(B) the application of biotechnology is driven by profits and the
promise of profits
(C) the distinction between Basic and Applied Science is often
blurred
(D) Basic Science is nearly immediately applied in today’s
biotech fields
(E) Basic Science has not yet applied in any of today’s biotech
fields
29. The development of Biotechnology is:
(A)
(B)
(C)
(D)
(E)
driven by application
has been banned in Europe by governments in the EU
has disproven the Theory of Evolution
destroying medicine as we know it
finally leveling off
30. The process of creating genetically modified
organisms (GMOs):
(A) has been applied to salmon
(B) has been applied to crop plants
(C) has not been commercialized for beef
(D) has been not demonstrated in peer review journals to cause
health issues
(E) all of the answers shown are correct