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Synthetic Biology

Synthetic Biology. Lecture 2: Fundamentals of Synthetic Biology. Fundamentals. Basic Components Promoters, Ribosome Binding Sites, Coding Sequences, terminators, Plasmids Isolating components from nature Basic Devices Inverters, Switches and Memories. Promoters.

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Synthetic Biology

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  1. Synthetic Biology Lecture 2: Fundamentals of Synthetic Biology

  2. Fundamentals • Basic Components • Promoters, Ribosome Binding Sites, Coding Sequences, terminators, Plasmids • Isolating components from nature • Basic Devices • Inverters, Switches and Memories

  3. Promoters • Regulatory parts (also known as promoters) are those which provide binding regions for RNA polymerase, the enzyme which performs the act of transcription (the production of RNA from a DNA template)

  4. The Lac Promoter http://web.mit.edu/esgbio/www/pge/lac.html

  5. The Lac Promoter

  6. The Lac Promoter

  7. The Lac Promoter

  8. Zinc Finger Promoters

  9. Harnessing ZFPs

  10. Ribosome Binding Sites • “Landing Site for Ribosomes” • Approximately 10 nt away from AUG

  11. RBS Binding

  12. RBS Manipulation • Adjust melting temperature of the Shine-Delgarno sequence • Add secondary structures to alter binding

  13. RBS Manipulation http://www.nature.com/nbt/journal/v22/n7/images/nbt986-F1.gif

  14. Coding Sequences • Code for a protein http://molvis.sdsc.edu/atlas/morphs/lacrep/lacrep_anim_small.gif

  15. Codon Usage Triplets (codons) of DNA/RNA code for amino acids Organisms ‘prefer’ different codons Re-coding amino acids can result in improved or reduced translation http://www.g-language.org/data/haruo/codon_table.gif

  16. Terminators • Forward and Reverse • BBa_B0025 http://parts.mit.edu/registry/index.php/Part:BBa_B0025

  17. Terminator Efficiency • Single terminators - • Forward and reverse efficiency • Current range -1.09 to .984 • Negative means it acts as a promoter • Terminators can be combined (B0021=B0010+B0012)

  18. Plasmids • Circular pieces of DNA that hold our devices • Origin of Replication • Copy Number • Antibiotic Resistance • Multiple-Cloning Site/BioBrick Insertion Site

  19. About Plasmids http://parts.mit.edu/registry/index.php/Help:Plasmid_features

  20. BioBrick Plasmids • Different Origins of Replication Required! • pSB1AK3 • [pSB] plasmid Synth Bio • [1] origin of Replication • [AK] Resistance (Amp/Kan) • [3] Version • Postfixed data is the insert • See http://parts.mit.edu/registry/index.php/Help:Plasmids/Nomenclature

  21. Plasmid-Plasmid Interactions

  22. Taming Nature • Most parts are derived from natural systems

  23. Building Devices • Devices are themselves parts, but they are built from several smaller components. • The choice of input/output of a device is very important, as it determines how parts can be ‘connected’.

  24. The Quad Part Inverter

  25. Features of QPI’s • Inverters work well because they are non-linear, and thus they are ‘restorative’.

  26. The QPI Abstraction Barrier

  27. Using Proteins as Signals

  28. Wait a sec… IF we use proteins as our signal carrier, we need to have inverters that handle all sorts of input/output combinations!

  29. Keep the protein self contained

  30. PoPS PoPS-> ->PoPS Polymerase Per Second

  31. Building a System Description

  32. Timing Diagram

  33. Drill down to Parts

  34. DNA Layout

  35. Add Debugging Parts

  36. Standard Assembly • Collect List of Devices to build, and build an assembly tree. • “Push Button” Synthesis • Automated Assembly means you have more time to test alternatives, test the resulting devices, and design more.

  37. Case: Repressilator

  38. An Oscillator

  39. Actual Behavior is Stochastic

  40. System Sensitivity to Parameters

  41. Plasmid Layout

  42. They Oscillate.. Sort of.

  43. Major Issues Raised • Load on Cells • Stochastic Variation in performance • Genetic Stability over time

  44. Load • How many cellular resources does the device use? • dNTPs (Marginal DNA replication) • rNTPs (RNA Production) • RiPS (Ribosomes) • Amino Acids (Proteins) • ATP for activity

  45. dNTP Load • Computation based on copy number and device length in nucleotides ldNTP= ncopy*lpart

  46. RNA Load • RiPS Usage: • Transcript count(production rate & stability), protein synthesis time • dN/dt = P-N*D • Assume synthesis time is proportional to transcript length t=a*l • NTP usage =N*l

  47. Amino Acids • Amino Acids • Protein length, copies • A=Ntranscripts*lprotein • N=Transcript length, l= protein length

  48. ATP (energy) • Demand is proportional the weighted sum of the other demands E=∑( aLDNA+bLRNA+cLAA ) Over all parts, plus the ATP required for coding sequence function.

  49. Dealing with Load • Need engineered chasses • Reduced genome organisms (mycoplasma) • Eliminate key components: recombinases, create dependencies, unnecessary parts.

  50. Can we win?

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