igem : international genetically engineered machine
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iGEM : International Genetically Engineered Machine. Team Members: Ashlee Smith, Emily Sileo, Clay Swackhamer, and Sam Krug. Overview. What is iGEM? DNA Fundementals Our Project Burning Issues: Why does this matter? Want to learn more?. iGEM. Teams from different schools - PowerPoint PPT PresentationTRANSCRIPT
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>.