🧬Systems Biology Unit 14 – Synthetic Biology: Circuit Design Basics

What's Synthetic Biology All About?

• Synthetic biology combines principles from biology, engineering, and computer science to design and construct new biological systems and functions
• Aims to create novel biological parts, devices, and systems that do not exist in the natural world
• Utilizes the power of genetic engineering and molecular biology techniques to modify or create organisms with desired traits
• Draws inspiration from electrical engineering and computer science to design biological circuits and programs
• Potential applications span various fields including medicine (drug development, gene therapy), agriculture (crop improvement), environmental remediation, and biomanufacturing (production of chemicals, materials)
• Raises ethical and safety concerns regarding the creation of artificial life forms and the potential unintended consequences of releasing engineered organisms into the environment
• Requires interdisciplinary collaboration among biologists, engineers, computer scientists, and other experts to tackle complex challenges

Key Players: Genes, Proteins, and Circuits

• Genes are the fundamental units of heredity that encode instructions for making proteins
  • Composed of DNA sequences that specify the amino acid sequence of proteins
  • Can be manipulated through genetic engineering techniques (gene insertion, deletion, modification)
• Proteins are the functional molecules that carry out various tasks in living organisms
  • Serve as enzymes catalyzing biochemical reactions, structural components, signaling molecules, and regulators of gene expression
  • Protein function is determined by its amino acid sequence and three-dimensional structure
• Genetic circuits are networks of interacting genes and proteins that perform specific functions
  • Analogous to electronic circuits where genes and proteins act as switches, sensors, and actuators
  • Can be designed to respond to specific inputs (small molecules, light) and produce desired outputs (fluorescent proteins, metabolites)
• Understanding the interplay between genes, proteins, and circuits is crucial for designing functional biological systems
• Synthetic biologists aim to create standardized, modular, and predictable genetic parts and circuits that can be easily assembled and optimized

Building Blocks of Genetic Circuits

• Promoters are DNA sequences that initiate transcription of genes
  • Serve as binding sites for RNA polymerase and transcription factors
  • Can be constitutive (always active) or inducible (activated by specific signals)
• Ribosome binding sites (RBS) are RNA sequences that recruit ribosomes for translation of mRNA into proteins
  • Determine the efficiency of protein synthesis
• Coding sequences (CDS) are DNA sequences that encode the amino acid sequence of proteins
  • Can be optimized for expression in different host organisms (codon optimization)
• Terminators are DNA sequences that signal the end of transcription
  • Prevent read-through into adjacent genes
• Operators are DNA sequences that bind transcription factors to regulate gene expression
  • Can be activating (enhancing transcription) or repressing (blocking transcription)
• These building blocks can be combined in various ways to create complex genetic circuits with desired functions
  • Promoters and RBS can be used to control the strength and timing of gene expression
  • CDS can be swapped to produce different proteins with specific functions
  • Operators can be used to create feedback loops and regulatory networks

Logic Gates in Biology: AND, OR, NOT

• Logic gates are fundamental building blocks of electronic circuits that perform Boolean logic operations
• Synthetic biologists have created analogous genetic logic gates using genes and proteins
• AND gate: Output is produced only when all inputs are present
  • Can be implemented using a promoter that requires multiple transcription factors to bind for activation
• OR gate: Output is produced when at least one input is present
  • Can be implemented using multiple promoters that independently drive expression of the same output gene
• NOT gate: Output is produced only when the input is absent
  • Can be implemented using a repressor protein that blocks transcription in the presence of an input signal
• Combining these basic logic gates allows the creation of more complex circuits (XOR, NAND, NOR)
• Genetic logic gates can be used to process multiple inputs and make decisions based on environmental conditions
  • Example: A circuit that produces a therapeutic protein only in the presence of two disease biomarkers

Designing Your First Genetic Circuit

• Define the desired function and inputs/outputs of the circuit
  • What task should the circuit perform? What signals will it respond to and what will it produce?
• Select appropriate genetic parts (promoters, RBS, CDS, terminators) based on the design requirements
  • Consider factors such as host organism, expression levels, and compatibility between parts
• Assemble the parts into a complete circuit using DNA assembly methods (restriction enzymes, Gibson assembly, Golden Gate assembly)
  • Ensure proper orientation and spacing of genetic elements
• Introduce the circuit into a suitable host organism (bacteria, yeast, mammalian cells) for testing
  • Use plasmid vectors or genomic integration methods
• Characterize the performance of the circuit using various assays (fluorescence, enzymatic activity, metabolite production)
  • Measure input-output relationships, dynamic behavior, and robustness
• Optimize the circuit based on the characterization data
  • Fine-tune expression levels, replace underperforming parts, and introduce additional regulation
• Validate the functionality of the optimized circuit in the intended application context
  • Test in more complex environments (co-cultures, animal models) and assess safety and biocontainment

Tools and Techniques for Circuit Assembly

• Restriction enzymes are used to cut DNA at specific recognition sites
  • Allows the creation of compatible sticky ends for ligation of parts
• Gibson assembly is a method for assembling multiple DNA fragments in a single reaction
  • Uses overlapping sequences and a combination of enzymes (exonuclease, DNA polymerase, DNA ligase)
• Golden Gate assembly is a method for assembling multiple parts using type IIS restriction enzymes
  • Enables scarless assembly and standardized modular cloning
• Polymerase chain reaction (PCR) is used to amplify specific DNA sequences
  • Allows the introduction of modifications (mutations, restriction sites) and the creation of fusion proteins
• DNA synthesis is used to chemically synthesize oligonucleotides and longer DNA fragments
  • Enables the creation of novel sequences and codon optimization
• Genome editing tools (CRISPR-Cas9, TALENs, zinc finger nucleases) are used to make targeted modifications to genomes
  • Allows the integration of circuits into specific genomic loci and the creation of knockout strains
• Bioinformatics tools (sequence analysis, modeling, circuit design software) are used to aid in the design and optimization of genetic circuits
  • Helps predict circuit behavior, identify potential issues, and guide experimental design

Challenges and Troubleshooting

• Biological complexity and context-dependence can make circuit behavior unpredictable
  • Genetic background, metabolic state, and environmental factors can influence circuit performance
• Incomplete understanding of biological systems can lead to unexpected interactions and off-target effects
  • Unintended cross-talk between circuit components and host pathways can disrupt function
• Evolutionary instability can cause circuits to mutate and lose function over time
  • Selection pressures can favor the inactivation of burdensome or toxic circuit elements
• Insufficient characterization and standardization of genetic parts can hinder reproducibility and scalability
  • Variability in part performance across different contexts can make circuit design challenging
• Limited host range and compatibility issues can restrict the applications of genetic circuits
  • Some circuits may not function properly in industrially relevant organisms or complex environments
• Addressing these challenges requires a combination of improved biological understanding, robust design principles, and rigorous testing and validation
  • Iterative design-build-test cycles and the incorporation of feedback control can help mitigate some of these issues

Real-World Applications and Future Directions

• Metabolic engineering: Genetic circuits can be used to optimize the production of valuable compounds (drugs, fuels, materials) in microorganisms
  • Example: Engineered yeast strains that produce high levels of artemisinic acid, a precursor to the antimalarial drug artemisinin
• Biosensing and diagnostics: Genetic circuits can be designed to detect specific molecules or conditions and produce a measurable output
  • Example: A paper-based synthetic gene network that detects Ebola virus RNA and produces a color change
• Bioremediation: Engineered microbes with genetic circuits can be used to degrade pollutants or sequester heavy metals from the environment
  • Example: Bacteria engineered to sense and degrade oil spills in marine environments
• Therapeutics: Genetic circuits can be used to create smart therapeutics that sense disease states and deliver targeted treatments
  • Example: Engineered immune cells (CAR-T cells) that recognize and kill cancer cells
• Agriculture: Genetic circuits can be used to create crops with improved traits (resistance to pests, drought tolerance) or to control plant growth and development
  • Example: A synthetic genetic circuit that regulates the timing of flowering in plants
• Future directions in synthetic biology include the development of more complex and sophisticated circuits, the integration of multiple circuits into larger systems, and the exploration of novel applications in areas such as materials science, data storage, and artificial life
  • The field will continue to benefit from advances in DNA synthesis, genome editing, and computational tools for circuit design and modeling