Synthetic biology: basic concepts • Synthetic biology follows a hierarchicla structure, building up systems from smaller components. Synthetic biology is in many aspects similar to electric engineering: cellular decision-making processes share basic operations with electronic control circuits. Intra- and extracellular information is collected by sensors that communicate the input signal states into a network, which processes the data according to logic and arithmetic operations. These operations result in decisions that are finally executed by output signals. • An important aspect of synthetic biology is the application of systemic design. This approach is based on the engineering principles of modularity, characterization and standartization. Synthetic biology: abstraction hierarchy and modularity Engineering Synthetic Biology Parts Devices Systems protein promoter miRNA Inducible protein expression module Synthetic therapeutic program to specifically kill a cancer cell Basicbiological functions Combinationofparts performingahuman definedfunction Combinationof devices Tools (parts) for regulating gene expression Xiang et al., Natural Comp 2018 Parts • Promoters: recruits RNA polymerase and other accessory proteins to prime the transcription of mRNA. Promoters have different strength and can be constitutive or regulated (inducible, repressible). • Protein coding sequences: encode transcriptional activators and repressors, transcription factors, sensors (e.g. cryptochromes for light perception, receptors for chemical ligands), signaling molecules (kinases, proteases), protein scaffolds and „output“ proteins (antibiotic resistance, GFP, LUX, suicidal proteins). • Teminators: signal termination of transcription, polyA signals. • Factors affecting RNA stability: bacterial mRNA has very short half life (average 2 min in E.coli). In contrast, stability of eukaryotic mRNA can range form minutes to days. Cis-elements affecting RNA stability: length of 3´UTR, hairpins, RNA binding motives, introns. • Ribosome binding sites: recruit ribosome for initiation of translation (Shine-Delgarno nad Kozak sequences). These sequences affect efficiency of transaltion and hence protein production. • siRNAs, miRNAs: affect gene expression on postrancriptional or translational level. • Translational riboswitches: a regulatory segment within a mRNA that can bind a small molecule, which in turn affects translation (eg lysisn riboswitch). Orthogonality in synthetic biology Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal (non-interferig) to the host background and to other synthetic networks. It also implies context independent performance of a synthetic network. TetR repressor • Relatively short operator sequence • Often used in synthetic genetic circuits • Thousands of homologues that exhibit binding specificities to disparate operators are available Stanton et al, Nat Chem Biol 2014 Mining of prokaryotic genomes for orthogonal TetR repressors Gene Bank: 82,017 TetR reprssors 73 preselected for experimental characterization 2.1 Mio 28 bp inverted repeats screened on microarrays using Cy5 labeled TetRs 16 operator-repressor pairs that exhibit strong repression and minimal corss-talk with other promoters/repressors Synthetic transcription regulators based on TALE and CRISPR/Cas9 Mercer et al, ACS Synt Biol 2013 Devices Tetracycline regulated siRNA production Tetracycline regulated protein production via proteolytic cleavage • Combination of parts performing a human defined function. Devices Auxin inducible degron Red light regulated expression Synthetic logic gates and cellular computation A logic gate in electronics, is a physical device which is implemented with a Boolean function based on input and output signals (0 and 1). It executes a logical function on one or more inputs that produce a single output. Logic gates are used for storing the data that can be constructed by connecting several gates in a Flip-flops circuit which is a central building block of digital electronics systems in computers and communications. In biological systems, logic gates are synthetic gene circuits programmed to permit the expression of an output protein only when a strictly defined signature of input signals is matched. Genetic elements interact with regulatory proteins to switch a gene ON or OFF while RNA or protein concentration can serve as input or output. One of the key approaches of synthetic biology is to reprogram the decision-making gene networks in order to implement them as logic gates in living systems. The AND gate gives a high output (1) only if both the inputs are high. Singh, Syst Synth Biol 2014 Logic gates: the AND gate The NOT gate module The modular NOT gate was designed on the basis of the cI/Plam repressor module consisting of lambda gene cI and its regulatory PR promoter. Wang et al., Nat Comm 2011 • A NOT gate takes a single input and inverts it, so 0 becomes 1; 1 becomes 0. • Even these simple gates can perform signal-processing functions, for example, converting a dark sensor into a light sensor. The NAND gate The output of NAND gate is high if any of inputs are low. This NAND gate results from the combination of AND and NOT gates. The AND gate is derived from hetero-regulation module from Pseudomonas syringae. The device comprises two coactivating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed. Wang et al., Nat Comm 2011 An OR gate outputs 1 as long as either (or both) of the inputs is 1. The OR gate A NOR gate is equivalent to OR gate followed by a NOT gate. The outputs of NOR gates are low, if any of the inputs are high. The NOR gate An exclusive-OR (XOR) gate gives a high output only if either input is present. The XOR gate Wong et al., BMC Biology 2015 Xie and Fussenegger, NatRevMCB 2018 Assembly of logic gates allows building circuits with complex behaviour A multilayer AND gate Singh, Syst Synth Biol 2014 Toggle switch and repressilator Toggle switch Repressilator Relaxation oscillator uses autoregulatory feedback Negative feedback loop rescues the noice and results in a narrow expression distribution Registry of parts for synthetic biology The basic concept in engineering is using a combination of standart parts to produce standard devices, which are then combined to produce standard systems. • Standart parts must be thoroughly characterized and their performance well described • Data on standartized parts are organized in registries of parts for synthetic biology MIT registry for the International Genetically Engineered Machine (iGEM) http://parts.igem.org/Main_Page • iGEM Registry provides a resource of available biological parts that have been user-tested and characterized for users developing synthetic biology projects. • iGEM Registry is an open community that runs on the "Get & Give & Share" philosophy. Users get parts, samples, data, and tools to work on their synthetic biology projects. They'll give back to the Registry the new parts they've made, as well as data and experience on new and existing parts. • The parts on the iGEM Registry adhere to the BioBrick standard allowing them to be assembled together creating new longer and more complex parts, while still maintaining the structural elements of the standard. This allows the engineer to focus on design instead of assembly. • BioBrick Assembly Standart 10 is based on restriction cloning • Other registries: SYNBis Database http://synbis.bg.ic.ac.uk/synbis2/Welcome_Page.html • iGEM Registry contains about 20,000 parts Standartization: The Synthetic Biology Open Language (SBOL) • SBOL is an open standard for the representation of in silico biological designs. • SBOL also provides schematic glyphs to graphically depict genetic designs called SBOL Visual. https://sbolstandard.org/ The synthetic biology design cycle Specification: formal definition of the desired function and design of a target genetic system. Design: the set of decisions needed to determine the constructs and hosts, to be used and/or modifications to the host to be made. Involves also creating a plan for composing the DNA constructs from their elements. Build: Implementing DNA assembly plan and construction of the biological system. Test: design and implementaiton of experiments for characterizing engineered systems and accompanying analysis and data interpretation. Learn: include approaches to allow for revision of designs based on experimental outcomes. • BioCAD assists the de novo design and selection of existing genetic components to achieve a desired biological activity, as part of an integrated designbuild-test cycle. Biological Computer Aided Design (BioCAD) Lux et al., TiBiotech 2012 • BioCAD tools facilitate the design of larger systems from smaller genetic parts by providing users with visual, textual or programming-language-like interfaces, or automatically generating designs from intended function. • As the field moves towards real-world applications, tools that can adequately predict functionality from design will be indispensable. • Predictability of part behavior, particularly in different contexts, is still a major issue in synthetic biology design. Nielsen et al., Science 2016 Recommended reading: Wang et al. (2011) Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat. Comm 1:508 Xiang et al. (2018) Scaling up genetic circuit design for cellular computing: advances and prospects. Natural Comp. 4:833