Synthetic Biology

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Synthetic Biology

• Lecture 2 Fundamentals of Synthetic Biology

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Fundamentals

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

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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)

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The Lac Promoter
http//web.mit.edu/esgbio/www/pge/lac.html
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The Lac Promoter
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The Lac Promoter
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The Lac Promoter
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Zinc Finger Promoters
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Harnessing ZFPs
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Ribosome Binding Sites

• Landing Site for Ribosomes
• Approximately 10 nt away from AUG

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RBS Binding
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RBS Manipulation

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

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RBS Manipulation
http//www.nature.com/nbt/journal/v22/n7/images/nb
t986-F1.gif
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Coding Sequences

• Code for a protein

http//molvis.sdsc.edu/atlas/morphs/lacrep/lacrep_
anim_small.gif
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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.g
if
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Terminators

• Forward and Reverse
• BBa_B0025

http//parts.mit.edu/registry/index.php/PartBBa_B
0025
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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 (B0021B0010B0012)

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Plasmids

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

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About Plasmids
http//parts.mit.edu/registry/index.php/HelpPlasm
id_features
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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/HelpP
  lasmids/Nomenclature

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Plasmid-Plasmid Interactions
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Taming Nature

• Most parts are derived from natural systems

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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.

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The Quad Part Inverter
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Features of QPIs

• Inverters work well because they are non-linear,
  and thus they are restorative.

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The QPI Abstraction Barrier
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Using Proteins as Signals
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Wait a sec

• IF we use proteins as our signal carrier, we need
  to have inverters that handle all sorts of
  input/output combinations!

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Keep the protein self contained
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PoPS
-gtPoPS
PoPS-gt
Polymerase Per Second
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Building a System Description
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Timing Diagram
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Drill down to Parts
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DNA Layout
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Add Debugging Parts
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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.

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Case Repressilator
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An Oscillator
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Actual Behavior is Stochastic
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System Sensitivity to Parameters
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Plasmid Layout
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They Oscillate.. Sort of.
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Major Issues Raised

• Load on Cells
• Stochastic Variation in performance
• Genetic Stability over time

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Load

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

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dNTP Load

• Computation based on copy number and device
  length in nucleotides
• ldNTP ncopylpart

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RNA Load

• RiPS Usage
• Transcript count(production rate stability),
  protein synthesis time
• dN/dt P-ND
• Assume synthesis time is proportional to
  transcript length tal
• NTP usage Nl

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Amino Acids

• Amino Acids
• Protein length, copies
• ANtranscriptslprotein
• NTranscript length, l protein length

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ATP (energy)

• Demand is proportional the weighted sum of the
  other demands
• E?( aLDNAbLRNAcLAA )
• Over all parts, plus the ATP required for coding
  sequence function.

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Dealing with Load

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

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Can we win?