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
The Lac Promoter
Zinc Finger Promoters
Harnessing ZFPs
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
Coding Sequences Code for a protein
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
Terminators Forward and Reverse BBa_B0025
Terminator Efficiency Single terminators - –Forward and reverse efficiency –Current range 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
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
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
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 ->PoPSPoPS-> Polymerase Per Second
Building a System Description
Timing Diagram
Drill down to Parts
DNA Layout
Add Debugging Parts
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
Actual Behavior is Stochastic
System Sensitivity to Parameters
Plasmid Layout
They Oscillate.. Sort of.
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 l dNTP = n copy *l part
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 –Protein length, copies –A=N transcripts *l protein –N=Transcript length, l= protein length
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.
Dealing with Load Need engineered chasses –Reduced genome organisms (mycoplasma) –Eliminate key components: recombinases, create dependencies, unnecessary parts.
Can we win?