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iGEM : International Genetically Engineered Machine

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

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iGEM : International Genetically Engineered Machine

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  1. iGEM:International Genetically Engineered Machine Team Members: Ashlee Smith, Emily Sileo, Clay Swackhamer, and Sam Krug

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

  3. iGEM • Teams from different schools • Genetically engineer something that will benefit society • BioBrick registry • Lots of FUN!!!

  4. DNA: Basics • 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.

  5. DNA: Basics • 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

  6. DNA and RNA: What is the Difference? • DNA • A,T,C,G • Double Stranded • RNA • A,U,C,G • Single Stranded • Different types • mRNA • tRNA • rRNA

  7. A closer look at transcription • DNA is “unzipped” • Promoter tells enzymes where to start coding • Oncetranscription is done, DNA returns to double strand and mRNA leaves the cell

  8. A closer look at translation • Ribosomes – the place where translation occurs • tRNA • Brings amino acids to the mRNA chain. • Many amino acids together is called a polypeptide chain

  9. Genes – Not the ones you wear • Segments of DNA • Are associated with a specific protein • Environmental factors can influence • https://www.youtube.com/watch?v=oBwtxdI1zvk

  10. Activity: Central Dogma Relay

  11. Our Project Bacteria generate useful compounds

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

  13. Some Solutions • Non-Genetic Level: Influence growth rate, extraction rate, purification efficiency • Genetic Level: Control output of product (expression)

  14. Why Do We Need a Third Control? • Engineers want more control over their systems • Increase output • In case of failure

  15. What are these controls? Ribosome Binding Site Promoter

  16. We are adding a third knob Ribosome Binding Site Codon Optimization Promoter

  17. How does it work? • 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

  18. Synonymous Codons UUU Phenylalanine UUC

  19. But are Synonymous Codons Exactly the Same?

  20. Maybe One Adds the Lego Faster?

  21. In Actual Biology • We know the codons in many genes • We have the ability to rebuild the genes using only the codons we want

  22. Example AUU AUC Isoleucine AUA

  23. Putting it together… AUU AUC AUA Old Gene: Old Amino Acid Sequence: New Gene: AUU AUU AUU New Amino Acid Sequence:

  24. But how do we know which codons to pick? • Complicated

  25. How do we actually make the gene? • 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

  26. How do we see if it works? • Test it in living cells!

  27. The Red Arrow • 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

  28. What is a Plasmid? • Circular Piece of DNA • Serves as a shuttle for genes • May include non-wild type DNA • Viral • Synthetic • Random

  29. How do we get the plasmid into the cells? • Electroporation • Heat Shock

  30. But These are “Shotgun” Approaches… • No way to see right away which piece of DNA the cell took in • If it even got one…

  31. Reminder! • Plasmid is a tool to introduce our synthetic genes to living cells Plasmid carrying our gene

  32. Back to figuring out which cells got the DNA we wanted • Grow the cells on a substance that will kill them without a gene that is in the plasmid • Antibiotic plates

  33. Accelerated Evolution… • 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

  34. Plasmid Carries Information • We are not giving the cell a barrier, or antidote, or chemical to destroy the antibiotic

  35. Problem! • 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

  36. Relax… • 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

  37. We Need to Work on Our Plasmid… Parent Cell Daughter Cells

  38. How can we get the plasmid into all the cells?

  39. Origin of Replication Now all daughter cells have the plasmid!

  40. Back to our plasmid • 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 Selection Marker Origin of Replication

  41. Inserting the Gene • Use Restriction Enzymes • Takes advantage of a primitive bacterial immune system

  42. Transcription • Need a way to show cell where to start translating and where to stop Terminator Promoter

  43. Translation • Need a way to tell cell where to start translating and where to stop Stop Codon Ribosome Binding Site Start Codon

  44. Back to our Controls… Ribosome Binding Site Promoter

  45. Start and Stop Codons • 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

  46. Your turn!

  47. Ethics in Synthetic Biology • Burning Issues • Fighting Extinction • Could Save endangered species • Could result in biodiversity issues

  48. Burning Issues • Genetically Modified Food • Bad media connotations • Could help to feed countries • Could create new allergies for humans

  49. Burning Issues • Altering babies before birth • Could save children from medical conditions • Could be exploited to alter other genes, like eye color, short or tall, etc.

  50. Activity on Ethics

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