Transcript Synthetic Biology

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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
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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!
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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
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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
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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.
Case: Repressilator
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
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?