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Synthetic biology principles

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... two sites, one where it behaves as an activator and one ... domain to a new, heterologous signal transduction domain used to control a gene of interesting. ... – PowerPoint PPT presentation

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Title: Synthetic biology principles


1
Synthetic biology principles
2
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

3
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.

4
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

5
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) .
6
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.

7
  • 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

8
  • 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.

9
  • 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.

10
  • 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.
11
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.
12
  • 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.

13
  • 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
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