1 / 50

Fundamentals of Synthetic Biology: Basic Components and Devices

Explore the fundamentals of synthetic biology, including basic components like promoters, ribosome binding sites, coding sequences, terminators, and plasmids. Learn about isolating components from nature and building basic devices such as inverters, switches, and memories.

ericortiz
Download Presentation

Fundamentals of Synthetic Biology: Basic Components and Devices

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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?

More Related