Synthetic biology principles

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Synthetic biology principles
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Problems with biological design cycle
manipulation of parts that are not quantitatively
characterized with various operating contexts

• Genes and networks responsible for a broad array
  of microbial functions were indentified,
  understood, then exploited for technological
  benefit. Bacteria were engineered to produce
  commodity chemicals, pharmaceuticals, and fuels.
  Design cycle, however, is costly due to
• Unclear mechanisms of part or part-part function
  constructs fail to operate as desired
• Contextual (on DNA) influence part function
  varies with respect to DNA context
• Non-quantified part performance no I/O transfer
  function with respect context
• Interference functions take place in same
  confined space of the cell
• Selection engineered systems evolve away from
  desired function
• Even with characterized parts, behavior (transfer
  function) has a stochastic component
• Cell-cell variation
• Fluctuation due to small numbers of molecules
• Noise in transcription and translation
• Noise from upstream part affects downstream part

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Synthetic biology a parts-based biological
circuit design cycle, with parts that conform to
design and performance requirements.

• 1. Standardized biological parts (functions)
• Predictable, quantitative behavior with respect
  to context
• Descriptions that facilitate part re-use (i.e.
  datasheets)
• I/O, part operating context context, measured
  quantitative behavior
• Dynamic behavior (I/O response) and steady-state
  behavior (with respect to context)
• 2. Composition rules that specify how objects
  must be assembled into functioning systems
• Physical composition how parts physically
  connected (i.e. wire standards)
• Functional composition system w/ expected
  behavior, no unintended emergent properties. To
  support functional composition, part properties
  and operating context are documented.
• 3. Kind of parts
• - Specialty parts specific function that has
  evolved over billions of years.
• - Generic parts interconnect specialty parts to
  form complex and predicable new functions in
  cells. First, build parts families that control
  transcription, translation, and protein-protein
  interaction. These parts enable a predictable
  biological circuit design cycle.

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Properties of scalable (rational framework to
determine parts behavior and appropriateness in
any system) biological parts

• Scalable biological parts need to have the
  following properties
• Independence part functions independent of host
  circuitry
• Reliability part functions as intended
• - Independence
• - Robustness in the face of noise
• - Part energetic load on host understood and
  optimized so that it is not selected against
• Tunability make controlled adjustments to part
  function
• Orthogonality part functions independent of
  other same-functioning parts
• - Parts tuned to the point of non-interference,
  despite having same function
• 5. Composability Parts can be combined to
  produce predictable functioning circuit

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Biological part properties
Threats
Examples
Independence
System function independent of host nitrogen
fixing system works when transplanted into E.
Coli Part functions independent of adjacent
circuitry repressors affect unique promoters
Different plasmid ORFs can interfere with each
other
Reliability
Function preserved by non-rigid design use noise
as a source of reliability to hedge against
uncertainty in the environment
Energetically-draining design protected
energetically burdensome part protected against
selection (by mutants) with markers
If parts are responsive to resources required
for transcription, translation, and replication,
mutants outcompete engineered system (Canton)
Tunability
Change system design to alter performance RBS to
change translation efficiency or tune mRNA
degradation (Keasling) tune RBS to produce
switch with graded or bi-stable response
(Collins) make proteins that function
conditionally (Duber) tunable circuit (Voigt)
Orthogonal
Tune to the extent that part specificity is
changed RNA designed to produce orthogonal parts
families - translational lock systems that block
translation and can be unlocked by small
molecules.
Composability
Parts assembled with predictable emergent
behavior a linking element between ribozyme
(responsive to an aptamer small molecule that
inhibits self-cleavage) and mRNA results in a
composite part. The individual part function are
preserved, and when coupling result in an
emergent function (degradation of the mRNA
transcript) .
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Example synthetic biological parts

• Sensors means of cell information receiving
• Small molecule
• Two-component
• Enviornemnt inducible
• Aptamer
• Circuits means of cell information processing
• Switch
• Inverter
• Bi-phasic
• Toggle
• Riboswitch
• Logic
• Gates
• Dynamic circuits
• Pulse generator
• Time delay
• Actuators output of a circuit can control a
  natural or transgenic response.

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• Sensors
• Small molecule inducer passed through cell
  membrane and binds to regulatory proteins to
• turn on activator or off a repressor, leading to
  activation or depression of a promoter.
• Lac graded induction
• Tet intermediate
• Ara all or none (i.e strongly cooperative so no
  intermediate induction)
• Two component systems the homology of
  intracellular parts intracellular sensor domain
• and response regulator is exploited to re-wire
  the circuit. The extracellular sensing domain
• is fused to a new intracellular signal
  transduction domain. In the canonical signal
  transduction
• system, membrane bound sensor phosphorylates a
  response regulator, which bind promoter.
• Light
• UV
• Environment inducible systems
• Oxygen
• Temperature
• pH

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• Circuits
• Switch turn on gene expression once an input
  has crossed cut-in value
• Transcriptional activators
• Or post-transcriptional mechanisms
• (DNA modifying enzymes)
• Riboregulators
• Inverter reciprocal response to input
• Input promoter linked to expression of a
  repressor
• Bi-phasic small input turn on band
• A regulator binds to two sites, one where it
  behaves as an activator and one where it behaves
  as a repressor. Differential affinity results in
  a certain response with respect to regulator
  concentration. If high affinity for the
  activator site, then low concentration is
  required to activate expression and high
  concentration to repress.
• Toggle two repressors that cross-regulate each
  others promoter
• Changing state requires modifying expression of
  one of the repressors.
• Serves as a memory device because it latches into
  one state and large perturbation necessary to
  flip it into the other state.
• Riboswitch block translation
• Adds a hairpin to the transcript, which overlaps
  with the RBS and prevents ribosome binding. This
  hairpin is disrupted by the expression of
  regulatory RNA. Inhibition is overcome by
  expression of a small regulatory RNA.

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• Circuits
• Logic apply computational operation to convert
  inputs to one or more outputs
• rRNA and mRNA orthogonal pairs that result in
  protein function when both expressed
• Aptamers small molecule inputs regulate gene
  expression
• Dynamic circuits whereas other circuits (logics
  gates and switches) are defined by their
  steady-state transfer function, circuits can also
  generate a dynamic response.
• Challenges robust to environmental conditions
  and minimal cell-cell variation
• Cascades temporally order gene expression
• Incoherent feedforward input activates a
  repressor and they together influence a
  downstream promoter. This forms a pulse generator
  when the repressor is turned on slowly and
  strongly affects the downstream promoter. Thus,
  the input incites a strong output, which is
  rapidly damped by the repressor.
• Coherent feedforward input and regulator have
  same influence on a downstream promoter. Produces
  a time-delay, in which short input pulses do not
  activate circuit.

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• Actuators
• Suicide
• Bio-film link a UV controlled switch to a gene
  that induces bio-film formation
• Adhesion / invasion

Obtaining synthetic control over a complicated,
multigene function might require deconstruction
of the natural regulation and the use of
synthetic regulation to control the entire
system. A step towards this goal was recently
demonstrated by refactoring and synthesizing a
version of T7 bacteriophage, which was engineered
to contain simplified regulation.
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Function
Application
Quorum Sensing
Colored rings density-dependant expression of
various florescent proteins, results in a color
pattern.
Light sensing
It is possible to fuse an extra-cellular light
sensing domain to a new, heterologous signal
transduction domain used to control a gene of
interesting. In this case, that gene produces
black pigment.
Oxygen Dependence
Anaerobic inducible promoter can be used to
create bacteria that can invade cancer cells in
the low-oxygen tumor micro-environment.
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• Inducible systems and switches exhibit
• Activation threshold
• Cooperativity of transition
• Cell to cell variation
• What are the challenges associated with building
  a system
• Connecting parts with matching timing and dynamic
  range
• Need to tune performance characteristics of
  parts
• Rationally mutate a part (operator or RBS)
• Database of parameterized genetic parts
• Directed evolution random mutagenesis
• Functional composition
• Need large toolbox of standardized and
  parameterized parts, and then a simple
  theoretical techniques to understand how these
  parts will function together.
• Theoretical inner workings use statistical
  mechanics to link transfer function to the
  thermodynamics of transcription factor binding
• Empirical relationship used to engineer linkages
  rapid determination of transfer function at the
  cell level with micro-fluidic devices
• Question how were electrical parts
  standardized, and what theoretical techniques
  helped engineers understand how these parts
  functioned together.

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• Key areas for improvement
• Construction of new parts that can be easily
  interchanged
• Increasing understanding of how parts can be
  wired together
• Development of new computational design methods
• Standardized data sharing