Transcript Principles

Synthetic biology principles
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:
1.Unclear mechanisms of part or part-part function: constructs fail to operate as desired
2.Contextual (on DNA) influence: part function varies with respect to DNA context
3.Non-quantified part performance: no I/O transfer function with respect context
4.Interference : functions take place in same confined space of the cell
5.Selection: engineered systems evolve away from desired function
Even with characterized parts, behavior (transfer function) has a stochastic component:
1.Cell-cell variation
2.Fluctuation due to small numbers of molecules
3.Noise in transcription and translation
4.Noise from upstream part affects downstream part
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 proteinprotein interaction. These parts enable a predictable biological circuit design cycle.
Properties of scalable (rational framework to determine part’s
behavior and appropriateness in any system) biological parts
Scalable biological parts need to have the following properties:
1. Independence: part functions independent of host circuitry
2. 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
3. Tunability: make controlled adjustments to part function
4. 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
Biological part
properties
Independence
Reliability
Examples
System function independent of host:
nitrogen fixing system works when
transplanted into E. Coli ; Part functions
independent of adjacent circuitry: repressors
affect unique promoters
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
Threats
Different plasmid ORFs
can interfere with each
other
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
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.
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.
1. Lac: graded induction
2. Tet: intermediate
3. 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.
1. Light
2. UV
Environment inducible systems :
1. Oxygen
2. Temperature
3. pH
Aptamer: small RNA molecules that change conformation when bound to an input can
Circuits
Switch : turn on gene expression once an input has crossed “cut-in” value
1. Transcriptional activators
2. Or post-transcriptional mechanisms
1. (DNA modifying enzymes)
2. Riboregulators
3. Inverter : reciprocal response to input
1. Input promoter linked to expression of a repressor
4. Bi-phasic : small input turn on band
1. 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.
5. Toggle : two repressors that cross-regulate each other’s promoter
1. Changing state requires modifying expression of one of the repressors.
2. Serves as a memory device because it latches into one state and large perturbation
necessary to flip it into the other state.
6. 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.
Circuits
Logic: apply computational operation to convert inputs to one or more outputs
1. rRNA and mRNA : orthogonal pairs that result in protein function when both expressed
2. 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.
1. Challenges : robust to environmental conditions and minimal cell-cell variation
2. Cascades : temporally order gene expression
1. 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.
2. 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.
Actuators
1. Suicide
2. Bio-film – link a UV controlled switch to a gene that induces bio-film formation
3. 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.
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.
Inducible systems and switches exhibit :
1. Activation threshold
2. Cooperativity of transition
3. Cell – to – cell variation
What are the challenges associated with building a system :
1. Connecting parts with matching timing and dynamic range
1. Need to : tune performance characteristics of parts
2. Rationally mutate a part (operator or RBS)
3. Database of parameterized genetic parts
4. Directed evolution: random mutagenesis
2. Functional composition
1. Need large toolbox of standardized and parameterized parts, and then a simple
theoretical techniques to understand how these parts will function together.
1. Theoretical inner workings : use statistical mechanics to link transfer function
to the thermodynamics of transcription factor binding
2. Empirical relationship used to engineer linkages : rapid determination of
transfer function at the cell level with micro-fluidic devices
2. Question : how were electrical parts standardized, and what theoretical techniques
helped engineers understand how these parts functioned together.
Key areas for improvement
1. Construction of new parts that can be easily interchanged
2. Increasing understanding of how parts can be wired together
3. Development of new computational design methods
4. Standardized data sharing