Synthetic biology principles

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 desired2.Contextual (on DNA) influence: part function varies with respect to DNA context3.Non-quantified part performance: no I/O transfer function with respect context4.Interference : functions take place in same confined space of the cell5.Selection: engineered systems evolve away from desired function

Even with characterized parts, behavior (transfer function) has a stochastic component:1.Cell-cell variation2.Fluctuation due to small numbers of molecules3.Noise in transcription and translation4.Noise from upstream part affects downstream part

Problems with biological design cycle: manipulation of parts that are not quantitatively characterized

with various operating contexts

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.

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

Scalable biological parts need to have the following properties:1. Independence: part functions independent of host circuitry2. 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 against3. Tunability: make controlled adjustments to part function4. Orthogonality: part functions independent of other same-

functioning parts - Parts tuned to the point of non-interference, despite having same

function5. Composability: Parts can be combined to produce predictable

functioning circuit

Properties of scalable (rational framework to determine part’s behavior and appropriateness in

any system) biological parts

Biological part

propertiesThreats

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

Examples

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)Orthogona

lTune 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) .

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.

Example synthetic biological parts

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 induction2. Tet: intermediate3. 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. Light2. UV

Environment inducible systems : 1. Oxygen 2. Temperature 3. pH

Aptamer: small RNA molecules that change conformation when bound to an input can

regulate translation – if fused to antisense RNA that interferes with RBS (translation only

when bound to input) – or transcription – if fused to ribozyme, which can target mRNA.

1. Tunable: rational design to change form of transfer function 2. Specific: target sequence engineered with W-C base pairing

Circuits

Switch : turn on gene expression once an input has crossed “cut-in” value

1. Transcriptional activators2. Or post-transcriptional mechanisms

1. (DNA modifying enzymes)2. Riboregulators

3. Inverter : reciprocal response to input1. Input promoter linked to expression of a repressor

4. Bi-phasic : small input turn on band1. 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 promoter1. 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. Suicide2. Bio-film – link a UV controlled switch to a gene that induces bio-

film formation3. 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

QuorumSensing

Colored rings: density-dependant expression of various florescent proteins, results in a color pattern.

Application

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.

OxygenDependen

ce

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 threshold2. 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 composition1. 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 improvement1. Construction of new parts that can be easily interchanged2. Increasing understanding of how parts can be wired together3. Development of new computational design methods4. Standardized data sharing