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

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 ->PoPSPoPS-> 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 l dNTP = n copy *l part

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 –Protein length, copies –A=N transcripts *l protein –N=Transcript length, l= protein length

48. ATP (energy) Demand is proportional the weighted sum of the other demands E=∑( aL DNA +bL RNA +cL AA ) 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?