QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 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 • 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) The Lac Promoter http://web.mit.edu/esgbio/www/pge/lac.html The Lac Promoter The Lac Promoter The Lac Promoter Zinc Finger Promoters Harnessing ZFPs QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Ribosome Binding Sites • “Landing Site for Ribosomes” • Approximately 10 nt away from AUG RBS Binding RBS Manipulation • Adjust melting temperature of the Shine-Delgarno sequence • Add secondary structures to alter binding RBS Manipulation http://www.nature.com/nbt/journal/v22/n7/images/nbt986-F1.gif Coding Sequences • Code for a protein http://molvis.sdsc.edu/atlas/morphs/lacrep/lacrep_anim_small.gif 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 Terminators • Forward and Reverse • BBa_B0025 http://parts.mit.edu/registry/index.php/Part:BBa_B0025 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) Plasmids • Circular pieces of DNA that hold our devices • Origin of Replication • Copy Number • Antibiotic Resistance • Multiple-Cloning Site/BioBrick Insertion Site About Plasmids http://parts.mit.edu/registry/index.php/Help:Plasmid_features 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 Plasmid-Plasmid Interactions Taming Nature • Most parts are derived from natural systems 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’. The Quad Part Inverter Features of QPI’s • Inverters work well because they are non-linear, and thus they are ‘restorative’. The QPI Abstraction Barrier Using Proteins as Signals QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Wait a sec… IF we use proteins as our signal carrier, we need to have inverters that handle all sorts of input/output combinations! Keep the protein self contained PoPS PoPS-> ->PoPS Polymerase Per Second Building a System Description QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Timing Diagram QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Drill down to Parts QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. DNA Layout QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Add Debugging Parts QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 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. Case: Repressilator An Oscillator QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Actual Behavior is Stochastic QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. System Sensitivity to Parameters QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Plasmid Layout QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. They Oscillate.. Sort of. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Major Issues Raised • Load on Cells • Stochastic Variation in performance • Genetic Stability over time Load • How many cellular resources does the device use? – dNTPs (Marginal DNA replication) – rNTPs (RNA Production) – RiPS (Ribosomes) – Amino Acids (Proteins) – ATP for activity dNTP Load • Computation based on copy number and device length in nucleotides ldNTP= ncopy*lpart 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 Amino Acids • Amino Acids – Protein length, copies – A=Ntranscripts*lprotein – N=Transcript length, l= protein length 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. Dealing with Load • Need engineered chasses – Reduced genome organisms (mycoplasma) – Eliminate key components: recombinases, create dependencies, unnecessary parts. Can we win?