Team Members: Ashlee Smith, Emily Sileo, Clay Swackhamer, and Sam Krug

What is iGEM? DNA Fundementals Our Project Burning Issues: Why does this matter? Want to learn more?

Teams from different schools Genetically engineer something that will

benefit society BioBrick registry Lots of FUN!!!

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.

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

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

Segments of DNA Are associated with

a specific protein Environmental

factors can influence

https://www.youtube.com/watch?v=oBwtxdI1zvk

Bacteria generate useful compounds

What are some ways that production of a chemical compound by microorganisms could be controlled?

Non-Genetic Level: Influence growth rate, extraction rate, purification efficiency

Genetic Level: Control output of product (expression)

Engineers want more control over their systems

Increase output

In case of failure

Ribosome Binding Site

Promoter

Ribosome Binding Site

Promoter Codon Optimization

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

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

AUU AUC AUA

Old Amino Acid Sequence:

New Gene: AUU AUU AUU

New Amino Acid Sequence:

Putting it together…Old Gene:

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

Test it in living cells!

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

Circular Piece of DNA

Serves as a shuttle for genes

May include non-wild type DNA◦ Viral◦ Synthetic◦ Random

Electroporation Heat Shock

No way to see right away which piece of DNA the cell took in◦ If it even got one…

Plasmid is a tool to introduce our synthetic genes to living cells

Plasmid carrying our gene

Grow the cells on a substance that will kill them without a gene that is in the plasmid

Antibiotic plates

Bacteria that have continual pressure from antibiotics develop resistance on their own◦ Natural selection◦ Only cells with resistance can reproduce

We are speeding this up by introducing the information they need to resist a specific antibiotic

We are not giving the cell a barrier, or antidote, or chemical to destroy the antibiotic

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

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!

Our plasmid can now replicate And we can tell which bacteria got the

plasmid Time to put in our gene!

Origin of Replication

Selection Marker

Spot to put in our gene

Use Restriction Enzymes◦ Takes advantage of a primitive bacterial immune

system

Need a way to show cell where to start translating and where to stop

Promoter

Terminator

Need a way to tell cell where to start translating and where to stop

Start Codon

Stop CodonRibosome

Binding Site

Ribosome Binding Site

Promoter

Can use them to turn off or on

Switches, not dials

Get stronger promoters or Ribosome Binding Sites, put them into the plasmid just like the Coding Sequence

Burning Issues◦ Fighting Extinction

Could Save endangered species

Could result in biodiversity issues

Genetically Modified Food◦ Bad media

connotations◦ Could help to feed

countries◦ Could create new

allergies for humans

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

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

pFTV

Existing GFP

Cla1

Sac1

Pst1

Forward Primer

Reverse Primer

Old GFP (Origional Superfolder GFP)

Sac1

Pst1

Cla1

Sac1

Pst1

Cla1

gBLOCK

Pst1

Cla1Leader Sequence

Xho1

GFP variation

Stop Codon

Homogenizes the first 60 bp of each GFP

Ensures that an accurate range of translation initiation is sampled

RBS

LeaderLeaderLeaderLeader

Leader

Variant GFP 1Variant GFP 2Variant GFP 3Variant GFP 4

Variant GFP 5

Leader Sequence

GFP variation

Stop Codon

Pst1

Sac1

Xho1

Cla1

Pst1

Sac1

Xho1

Cla1

dRBS orderedSac1

Pst1

dRBS

Leader Sequence

Variant GFP

Sequence of DNA which is transcribed to RNA

Location where Ribosome attaches

Allows translation to begin

Varies in strength (stronger site means more ribosomes on the mRNA)

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

“Strength” of RBS is measured in terms of how well translation is started

Translation Initiation Rate (TIR)

Pst1

Sac1

Xho1

Cla1

dRBS

Leader Sequence

Variant GFP

Depends on how firmly Ribosome can bind to mRNA

Translation initiation Calculated by software developed at Penn

State

Translation elongation is modified while translation initiation kept the same

Translation elongation becomes rate limiting step

Expression should grow as GFP efficiency increased and with higher TIR

Insulin Production in E. coli Clotting factors HGH Detect Heavy metals in drinking water Break down hydrocarbon pollution Hydrolyze Furfural

http://www.dnalc.org/view/15929-How-insulin-is-made-using-yeast.html

Books to read!

http://www.responsibletechnology.org/10-Reasons-to-Avoid-GMOs http://www.csa.com/discoveryguides/gmfood/overview.php http://www.nytimes.com/2014/02/24/opinion/genetically-modified-babies.html?_r=0 https://www.youtube.com/watch?v=oBwtxdI1zvk http://www.inukaleo.com/categories.php?U_Id=2 https://www.sciencenews.org/article/brain-reconstruction-hints-dinosaur-communication http://4.bp.blogspot.com/_qxe_WPY0C8U/TMLFQK8XlJI/AAAAAAAAABw/0JkcHKbJ48g/s1600/short+vs+tall.gif

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-can-rebuild-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-plasmids-vectors>.

Chloramphenicol. Digital image. Chloramphenicol. Wikimedia Foundation, Inc., 27 July 2014. Web. 28 July 2014. <http://en.wikipedia.org/wiki/Chloramphenicol>.