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

Biological part properties Threats IndependenceSystem 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 ReliabilityFunction 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) TunabilityChange 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) OrthogonalTune 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. ComposabilityParts 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 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 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 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 Quorum Sensing Colored rings: density-dependant expression of various florescent proteins, results in a color pattern. Application Light sensingIt 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