Team Members: Ashlee Smith, Emily Sileo,
Clay Swackhamer, and Sam Krug
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What is iGEM?
DNA Fundementals
Our Project
Burning Issues: Why does this matter?
Want to learn more?
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Teams from different schools
Genetically engineer something that will
benefit society
BioBrick registry
Lots of FUN!!!
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All living things have
DNA
There are four
nucleotides: Adenine,
Thymine, Cytosine,
Guanine. More
commonly referred to
as A, T, C, and G.
Unique shape --double helix.
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Central Dogma
◦ DNA---> RNA -->Protein
Transcription – When
the information stored
in DNA is used to
assemble a strand of
mRNA.
Translation – When
mRNA is read by
ribosomes and an
amino acid is assigned
to each group of three
(3) nucleotides
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DNA
◦ A,T,C,G
◦ Double Stranded
• RNA
– A,U,C,G
– Single
Stranded
– Different types
– mRNA
– tRNA
– rRNA
• DNA is “unzipped”
• Promoter tells
enzymes where to
start coding
• Once transcription
is done, DNA
returns to double
strand and mRNA
leaves the cell
• Ribosomes – the
place where
translation
occurs
• tRNA
– Brings amino
acids to the
mRNA chain.
– Many amino
acids together is
called a
polypeptide
chain
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Segments of DNA
Are associated with a
specific protein
Environmental
factors can influence
https://www.youtub
e.com/watch?v=oBwt
xdI1zvk
Bacteria generate useful
compounds
What are some ways that production of a
chemical compound by microorganisms could
be controlled?
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Non-Genetic Level: Influence growth rate,
extraction rate, purification efficiency
Genetic Level: Control output of product
(expression)
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Engineers want more control over their
systems
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Increase output
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In case of failure
Ribosome
Binding Site
Promoter
Ribosome
Binding Site
Promoter
Codon
Optimization
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A gene is made up of codons
64 codons
20 amino acids (building blocks of proteins)
Some amino acids are produced by more than
one codon
These codons are synonymous
UUU
Phenylalanine
UUC
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We know the codons in many genes
We have the ability to rebuild the genes using
only the codons we want
AUU
AUC
AUA
Isoleucine
Putting it together…
AUU AUC AUA
Old Gene:
Old Amino Acid Sequence:
New Gene:
AUU AUU AUU
New Amino Acid Sequence:
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Complicated
• Can’t pick out codons one at a time…the genes are
too long!
• Need to write a program to do it for us
• Send the output to a company with equipment to
synthesize long elements of double stranded DNA
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Test it in living cells!
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Bacterial cells can pick up DNA from their
surroundings
We need to make sure they pick up the DNA
that we want them to
Need to use a plasmid
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Circular Piece of DNA
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Serves as a shuttle for genes
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May include non-wild type DNA
◦ Viral
◦ Synthetic
◦ Random
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Electroporation
Heat Shock
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No way to see right away which piece of DNA
the cell took in
◦ If it even got one…
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Plasmid is a tool to introduce our synthetic genes
to living cells
Plasmid carrying our gene
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Grow the cells on a substance that will kill
them without a gene that is in the plasmid
Antibiotic plates
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Bacteria that have continual pressure from
antibiotics develop resistance on their own
◦ Natural selection
◦ Only cells with resistance can reproduce
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We are speeding this up by introducing the
information they need to resist a specific
antibiotic
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We are not giving the cell a barrier, or
antidote, or chemical to destroy the antibiotic
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Antibiotic Resistance Bacteria
“Each year in the United States, at least 2 million
people become infected with bacteria that are
resistant to antibiotics and at least 23,000 people die
each year as a direct result of these infections. Many
more people die from other conditions that were
complicated by an antibiotic-resistant infection.”
-source: http://www.cdc.gov/ Antibiotic Threats in the United
States, 2013
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Our bacteria have no pathogenicity
Our bacteria cannot survive outside of the lab
◦ No ability to manufacture leucine
• Chloramphenicol is no longer used as
a clinical antibiotic
Parent Cell
Daughter
Cells
Now all
daughter
cells have
the plasmid!
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Our plasmid can now replicate
And we can tell which bacteria got the plasmid
Time to put in our gene!
Spot to put in
our gene
Origin of
Replication
Selection
Marker
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Use Restriction Enzymes
◦ Takes advantage of a primitive bacterial immune
system
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Need a way to show cell where to start
translating and where to stop
Terminator
Promoter
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Need a way to tell cell where to start
translating and where to stop
Ribosome
Binding
Site
Stop
Codon
Start Codon
Ribosome
Binding Site
Promoter
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Can use them to turn off or on
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Switches, not dials
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Get stronger promoters or Ribosome Binding
Sites, put them into the plasmid just like the
Coding Sequence
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Burning Issues
◦ Fighting Extinction
 Could Save
endangered species
 Could result in
biodiversity issues
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Genetically
Modified Food
◦ Bad media
connotations
◦ Could help to feed
countries
◦ Could create new
allergies for humans
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Altering babies before
birth
◦ Could save children from
medical conditions
◦ Could be exploited to alter
other genes, like eye color,
short or tall, etc.
By Clay Swackhamer and Sam Krug
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Use pFTV as a vector
Inverse PCR to get construct and add
restriction sites
Introduce GFP variation using gBlocks and
restriction sites
Introduce the dRBS using restriction sites
Screen for different levels of fluorscence
Sequence
Cla1
Existing GFP
Forward
Primer
Pst1
pFTV
Reverse Primer
Sac
1
Pst1
Cla1
Sac1
Old GFP (Origional Superfolder
GFP)
Pst1
Cla1
Sac1
gBLOCK
Pst
1
Leader
Sequenc
e
Xho
1
GFP
variation
Stop Codon
Cla1
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Homogenizes the first 60 bp of each GFP
Ensures that an accurate range of translation
initiation is sampled
RB
S
Leade
r
Leade
r
Leade
r
Leade
r
Variant
1
Variant
2
Variant
3
Variant
4
Leade
r
Variant GFP
5
GFP
GFP
GFP
GFP
Leader
Sequenc
e
Pst
1
Xho
1
GFP
variation
Stop
Codon
Cla1
Sac
1
Leader
Sequenc
e
Variant
GFP
Xho
1
Pst
1
Cla1
Sac
1
dRBS ordered
dRBS
Sac1
Pst1
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Sequence of DNA which is transcribed to RNA
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Location where Ribosome attaches
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Allows translation to begin
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Varies in strength (stronger site means more
ribosomes on the mRNA)
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Sequence contains degenerate nucleotides
Ex)
CGTATGATACAAAGCMTTACCGCMCTGCAG
Presence of two M’s (A or C) means there are
2* 2 distinct sequences represented by this
dRBS
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“Strength” of RBS is measured in terms of how
well translation is started
Translation Initiation Rate (TIR)
Leader
Sequenc
e
Xho1
Cla1
Pst1
dRBS
Sac1
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Depends on how firmly Ribosome can bind to
mRNA
Translation initiation
Calculated by software developed at Penn
State
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Translation elongation is modified while
translation initiation kept the same
Translation elongation becomes rate limiting
step
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Expression should grow as GFP efficiency
increased and with higher TIR
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Insulin Production in E. coli
Clotting factors
HGH
Detect Heavy metals in drinking water
Break down hydrocarbon pollution
Hydrolyze Furfural
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http://www.dnalc.org/view/15929-How-insulin-is-made-using-yeast.html
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Books to read!
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http://www.responsibletechnology.org/10-Reasons-to-Avoid-GMOs
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http://www.csa.com/discoveryguides/gmfood/overview.php
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http://www.nytimes.com/2014/02/24/opinion/genetically-modified-babies.html?_r=0
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https://www.youtube.com/watch?v=oBwtxdI1zvk
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http://www.inukaleo.com/categories.php?U_Id=2
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https://www.sciencenews.org/article/brain-reconstruction-hints-dinosaur-communication
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http://4.bp.blogspot.com/_qxe_WPY0C8U/TMLFQK8XlJI/AAAAAAAAABw/0JkcHKbJ48g/s1600/short+vs+tall.gif
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E-coli-streptococci. Digital image. Http://wp.auburn.edu/. Wordpress, n.d. Web. 28 July 2014. <http://wp.auburn.edu/aww/did-you-
know/>.
Biofuel Fermentation Reactors. Digital image. Uabio.org. Bioenergy Association of Ukraine, 2012. Web. 28 July 2014.
<http://www.uabio.org/en/activity/alternative-digest>.
Bernd, Karen. Chemical Structure of Ethanol. Digital image.Http://www.bio.davidson.edu/. Davidson College, 2010. Web. 28 July 2014.
<http://www.bio.davidson.edu/people/kabernd/berndcv/lab/online_website2/ethanol.html>.
F (Phe). Digital image. The Biology Project: Biochemistry. University of Arizona, 2003. Web. 28 July 2014.
<http://www.biology.arizona.edu/biochemistry/problem_sets/aa/phenylalanine.html>.
Carr, Karen. Digital image. RNA Experiment. Portland State University, 2014. Web. 28 July 2014.
<http://scienceforkids.kidipede.com/biology/cells/doing/rna.htm>.
We Can Rebuild Him. Digital image. Veryhilarious.com. Very Hilarious.com, 2014. Web. 28 July 2014. <http://veryhilarious.com/we-canrebuild-him/>.
MATLAB: The Language of Technical Computing. Digital image. MacDiarmid Emerging Scientists Association. Mathworks, 2014. Web. 28
July 2014. <http://mesa.ac.nz/?page_id=771>.
What Is a Plamid? Digital image. What Is a Plasmid? Addgene.org, 2014. Web. 28 July 2014.
<https://www.addgene.org/tools/protocols/whatisaplasmid/>.
"Addgene: What Is a Plasmid?" Addgene: What Is a Plasmid? Addgene.org, 2014. Web. 28 July 2014.
<https://www.addgene.org/tools/protocols/whatisaplasmid/>.
Plasmid Map. Digital image. What Is a Plasmid? Addgene.org, 2014. Web. 28 July 2014.
<https://www.addgene.org/tools/protocols/whatisaplasmid/>.
Rajagopal, Indira. Figure 6.12. Digital image. Genome Organization Contd.Oregon State University, 2009. Web. 28 July 2014.
<http://oregonstate.edu/instruction/bi314/summer09/dnarep.html>.
F Replicates in Cytoplasm and Passed to Daughter Cells. Digital image. The Magical World of Bacteria and Phages. Miami University, n.d.
Web. 28 July 2014. <http://www.bio.miami.edu/dana/250/25008_7print.html>.
Bacterial Transformation: Electroporation. Digital image. Society for Mucosal Immunology. Http://www.socmucimm.org/, 2014. Web. 28
July 2014. <http://www.socmucimm.org/bacterial-transformation-electroporation/>.
Bacterium Cell Illustration of the Chromosome and Plasmid DNA . Digital image. Commonly Used Plasmids/Vectors. African Biosafety
Network of Expertise, 2010. Web. 28 July 2014. <http://www.nepadbiosafety.net/subjects/biotechnology/commonly-used-plasmidsvectors>.
Chloramphenicol. Digital image. Chloramphenicol. Wikimedia Foundation, Inc., 27 July 2014. Web. 28 July 2014.
<http://en.wikipedia.org/wiki/Chloramphenicol>.