Synthetic Cells, Design, Methods and Construction, Advanced Cell and Immune Therapies

Luke McLaughlin, Biotech Digital Marketer, Business Developer and Life Science Content Creator

Synthetic cells in biotechnology represent an innovative frontier where scientists design and construct cells or cell-like structures from scratch. These synthetic cells are designed to mimic the behavior of natural biological cells, performing specific functions such as protein synthesis, metabolism, and self-replication. The overarching goal is to use these cells in various applications, including medicine, research, and industry.

Design and Construction of Synthetic Cells

The design and construction of synthetic cells often start with the synthesis of a minimal genome. A minimal genome contains only the genetic information necessary for the survival and reproduction of the cell under laboratory conditions. The process generally involves the following steps:

Genome Design

Genome Design: Scientists use computational models to determine which genes are essential for life and can be included in the synthetic genome. This involves stripping down existing genomic data to the bare essentials and, sometimes, redesigning genes to optimize their functions.

Genome Minimization: This involves reducing the genome to its most essential genes only, removing non-essential genes, and possibly incorporating synthetic genes. The goal is to streamline cellular functions and reduce metabolic burden.

Essential Genes Identification: Identifying genes that are essential for cell survival and function is critical. This often requires extensive research and experimentation to determine which genes are necessary for life and which can be omitted or replaced.

Synthetic Pathways: Incorporating synthetic pathways that do not naturally occur in the organism to enable new functions or improve existing ones. This could include pathways for the production of pharmaceuticals, biofuels, or other valuable chemicals.

Orthogonality: Designing components that do not interfere with the natural cellular machinery but can function independently within the cell. This ensures that synthetic elements operate without disrupting the cell’s natural processes.

Biocontainment: Ensuring that synthetic cells cannot survive outside a controlled environment. This often involves creating dependency on synthetic amino acids or other compounds that aren't found in nature.

Stability and Robustness: The synthetic genome must be stable under different environmental conditions and during cell division. It should also maintain functionality over time without significant mutations.

Compatibility and Integration: The synthetic genome must be compatible with the host cell's existing machinery. This involves ensuring that gene expression levels are appropriate and do not disrupt the cell’s homeostasis.

Scalability: The design should be scalable, allowing for the production of cells in large quantities in a cost-effective manner. This is important for industrial applications of synthetic biology.

Ethical and Safety Considerations: Addressing ethical issues associated with creating synthetic life forms, including potential risks to natural ecosystems and human health. Rigorous safety assessments and regulatory compliance are crucial.

Computational Modeling and Simulation: Using computational tools to model and simulate the behavior of synthetic genomes before physical construction. This helps predict potential problems and optimize designs.

Standardization and Modular Design: Developing standardized, interchangeable parts that can be used across different synthetic biology projects. This modularity allows for easier troubleshooting, modification, and improvement of synthetic cells.

DNA Synthesis

DNA Synthesis: Once the genome is designed, the DNA is chemically synthesized. This can be a challenging process, especially for large genomes, as it requires assembling multiple short DNA sequences into a complete genome.

Current DNA synthesis technologies and methods

1. Phosphoramidite Method (Chemical Synthesis): This is the most common and established method for synthesizing short DNA sequences known as oligonucleotides. The process involves the sequential addition of nucleotide residues to a growing DNA chain, where each addition is protected by a phosphoramidite group. The method is highly efficient for generating oligonucleotides up to about 200 bases long.

2. Microarray-Based Synthesis: This technology allows for the simultaneous synthesis of a large number of different oligonucleotides on a solid surface. DNA sequences are built up in parallel on a microchip, enabling the production of thousands to millions of unique sequences at once, which are useful for applications like genome-wide experiments and large-scale synthetic biology projects.

3. PCR-Based Amplification: Often used to generate larger quantities of DNA from a small initial sample, PCR (Polymerase Chain Reaction) amplifies a specific DNA sequence using cycles of temperature changes and enzyme activity. While primarily an amplification method, PCR can also be used creatively to synthesize new DNA sequences through techniques like overlap extension PCR.

4. Enzymatic DNA Synthesis: This emerging technology uses template-independent DNA polymerases to synthesize DNA molecules. It represents a more natural approach compared to chemical synthesis, potentially allowing for longer and more complex DNA sequences to be constructed with fewer errors.

5. Terminal Deoxynucleotidyl Transferase (TdT) Synthesis: This enzyme adds nucleotides to the 3' end of a DNA molecule without needing a template. TdT is used in specialized applications, such as adding random or defined sequences to the ends of DNA strands in immunology and molecular biology research.

6. Gene Synthesis via Assembly of Overlapping Oligonucleotides: This method constructs entire genes from shorter, overlapping synthetic oligonucleotides. The oligos are designed to anneal to each other based on overlapping regions, and then are enzymatically ligated or assembled via PCR to form a full-length DNA sequence. This technique is essential for synthesizing complete genes and even whole genomes from scratch.

Sequence Design

Sequence Design: The DNA sequence must be carefully designed to encode the desired functions while avoiding unintended interactions or conflicts within the cell. This includes selecting promoter sequences, codon optimization for efficient protein expression, and avoiding sequences that might lead to genetic instability.

Accuracy and Fidelity: High fidelity in the synthesis process is crucial to ensure that the DNA sequence is produced without errors. This involves using reliable synthesis methods and thorough sequencing to confirm the accuracy of the synthetic DNA.

Scalability: The method of DNA synthesis must be scalable, allowing for the production of large quantities of DNA as needed. This is particularly important for industrial applications where large volumes of synthetic cells are required.

Cost-effectiveness: The synthesis process should be cost-effective to make the production of synthetic cells economically viable. Reducing the cost of oligonucleotide synthesis and assembly is a significant factor in this context.

Speed of Synthesis: The technology should allow for rapid synthesis of DNA to speed up the development and deployment of synthetic cells. Faster synthesis methods can significantly accelerate research and production cycles.

Chemical Stability and Purity: The synthesized DNA must be chemically stable and free from impurities that could interfere with its function once inside a cell. This involves using high-quality reagents and robust purification techniques.

Integration Capability: The synthesized DNA must be able to be efficiently integrated into the host cell’s genome or exist as a stable, extrachromosomal element. This might involve the use of vectors, integration sites that promote stable maintenance, or systems that avoid integration but ensure replication and inheritance.

Biosecurity and Safety: Ensuring that the synthetic DNA does not encode harmful functions or contribute to genetic instability is critical. Regulatory compliance and ethical considerations must be addressed to prevent misuse and to ensure that the synthetic DNA is safe for use in various environments.

Longevity and Stability in Expression: The synthetic DNA should maintain its stability over many generations, with consistent expression of its encoded functions. This requires careful design to avoid degradation or loss through cellular mechanisms like recombination or silencing.

Modular and Standardized Components: Using standardized and modular DNA parts can facilitate easier assembly, troubleshooting, and modification of synthetic genomes. This promotes interoperability and repeatability in synthetic biology projects.

Ethical Considerations: The synthesis of DNA for synthetic cells must be conducted with consideration for ethical issues, including potential impacts on the environment and biodiversity, as well as the broader implications of creating synthetic life forms.

Genome Transplantation

Genome Transplantation: The synthetic genome is then transplanted into a host cell that has had its DNA removed. The synthetic genome reprograms the host cell to create a new, synthetic organism whose cell functions are defined by the synthetic DNA.

Recipient Cell Compatibility: The recipient cell must be compatible with the transplanted genome. This involves ensuring that the cell machinery can effectively express genes from the new genome and that cellular systems such as membrane proteins and metabolic pathways are supportive of the new genetic material.

Efficiency of Genome Introduction: The method used to introduce the genome into the recipient cell must be efficient and reliable. Techniques might include electroporation, microinjection, or other methods of cell membrane penetration that minimize damage to both the genome and the cell.

Cell Viability Post-Transplantation: Maintaining cell viability after genome transplantation is crucial. The process should ensure minimal disruption to essential cell functions and promote rapid recovery and adaptation to the new genomic content.

Genomic Stability: The transplanted genome must remain stable within the host cell, maintaining its integrity without fragmentation or significant mutation over time. This stability is crucial for the sustainable function of synthetic cells.

Expression Compatibility: The synthetic genome should be designed such that its gene products (proteins and RNAs) are compatible with the host cell's machinery. This includes considerations for codon usage, promoter strength, and RNA stability.

Elimination of Native Genome: If the transplantation involves replacing the host's original genome, complete and reliable removal or inactivation of the native genome is necessary to prevent interference with the synthetic genome's functions.

Biocontainment and Safety: The synthetic cell must have built-in biocontainment features to prevent it from surviving outside of controlled environments, thereby ensuring environmental safety. This might include auxotrophic strains or genetic kill switches.

Functional Verification: After transplantation, the functionality of the synthetic cell must be verified through a series of tests to confirm that all intended operations are performing correctly, and that the cell can reproduce and maintain its synthetic genome through cell divisions.

Scalability: The genome transplantation technique should be scalable, enabling the production of multiple synthetic cells efficiently and cost-effectively. This is important for practical applications in research and industry.

Ethical and Regulatory Compliance: The process must adhere to ethical guidelines and regulatory standards, addressing concerns such as environmental impact, potential misuse, and the broader implications of creating and using synthetic organisms.

Genome Integration and Maintenance: Ensuring that the synthetic genome is properly integrated into the cellular architecture of the recipient cell or maintained episomally in a stable form that does not disrupt cell function.

Cellular Chassis

Cellular Chassis: The choice of a cellular chassis—i.e., the cell type that receives the synthetic genome—is crucial. This can be a bacterium, yeast, or even mammalian cells, depending on the intended function of the synthetic cell.

Creating a cellular chassis for synthetic biology involves various technologies and methods that enable the construction, modification, and optimization of cells to serve as platforms for engineering biological functions. Here’s a short list of current technologies and methods used in constructing cellular chassis:

1. CRISPR-Cas Genome Editing: This powerful tool allows for precise edits to the DNA of a host cell. It can be used to delete, insert, or modify genetic material in the chassis to optimize its function, eliminate unwanted traits, or introduce new capabilities. CRISPR-Cas is particularly valuable for creating custom modifications in the cellular chassis.

2. Transposon-Mediated Insertion: Utilizing mobile genetic elements known as transposons, this method can be used to randomly or specifically insert genes into a host genome. It is useful for generating libraries of genetic variants and for identifying beneficial mutations by insertional mutagenesis.

3. Synthetic Biology Toolkits: These consist of standardized, modular biological parts (such as BioBricks) that can be assembled to modify or build new biological systems within a cell. Toolkits can include promoters, coding sequences, and termination sequences, facilitating the modular design and assembly of biological functions in the chassis.

4. Tn-Seq (Transposon Sequencing): A high-throughput technique that combines transposon mutagenesis with deep sequencing. It allows for the systematic analysis of gene function within a chassis by identifying essential genes and those involved in specific phenotypes, which helps in chassis optimization.

5. Auxotrophic Strains: By creating or using strains that lack the ability to synthesize a vital nutrient, researchers can control the growth and survival of the chassis, ensuring containment and safety. This is crucial for biocontainment and for experiments requiring precise control over cellular growth.

6. Automated Strain Engineering Platforms: These platforms use automation and robotics to rapidly construct and test genetic modifications in cellular chassis. They enable high-throughput engineering, testing, and optimization of metabolic pathways and other synthetic biology constructs.

7. Cell-Free Systems: These are experimental systems that utilize cellular components (like enzymes and ribosomes) in the absence of cell walls and growth-related metabolic pathways. Cell-free systems are used for prototyping genetic circuits and producing proteins, and they can serve as simplified chassis for various synthetic biology applications.

8. Yeast Synthetic Genomes (Sc2.0): An advanced project where entire chromosomes of yeast are synthetically constructed, allowing unprecedented control over genetic elements and genome architecture. This project aims to create completely redesigned yeast cells that can serve as versatile chassis for industrial and research purposes.

Robustness and Stability: The chassis should be genetically stable and robust under various environmental conditions. It should maintain its integrity and functionality over time and under the stress of housing foreign or synthetic elements.

Genetic Simplifiability: The chassis should allow for easy genetic manipulation, including deletions, insertions, and modifications. This often involves a well-characterized genome with available tools for precise genetic engineering.

Minimal Metabolic Burden: The chassis should have a minimal metabolic burden to efficiently allocate resources to engineered pathways and functions rather than to its own survival and reproduction, making it more efficient in producing desired outputs.

Compatibility with Synthetic Systems: The chassis must be compatible with the synthetic pathways and components introduced. This includes the ability to express foreign proteins, replicate synthetic DNA, and accommodate modified biochemical pathways.

Growth and Culture Conditions: The organism should be easy to culture with minimal requirements for nutrients and other growth factors. It should also have a reasonably fast growth rate to facilitate experimental turnover and industrial production.

Biocontainment Features: The chassis should have inherent or engineered biocontainment features to prevent its survival outside of a controlled environment, reducing the risk of unintended ecological impacts.

Scalability: The ability to scale up the chassis for industrial or large-scale applications is crucial. This includes ease of cultivation in bioreactors and the ability to maintain stable production under scaled-up conditions.

Ethical and Safety Considerations: The selection and modification of a cellular chassis must comply with ethical standards and safety regulations, considering potential impacts on health and the environment.

Well-characterized Biology: The biological and physiological properties of the chassis should be well understood to predict and control its behavior when it is engineered with new functionalities.

Modularity and Standardization: The chassis should support a modular approach, allowing for the standardized integration of synthetic parts and systems. This facilitates easier troubleshooting, modification, and enhancement of synthetic cells.

Compatibility with Analytical Tools: The chassis should be amenable to standard analytical and diagnostic tools used in synthetic biology, allowing for easy monitoring and characterization of its functions and responses.

Customizability: The chassis should allow for customization according to the specific needs of the application, whether for producing pharmaceuticals, degrading pollutants, or any other designed function.

Designing and constructing synthetic cells

Designing and constructing synthetic cells involves a multidisciplinary approach, incorporating techniques from synthetic biology, systems biology, and bioengineering. Here’s a breakdown of the primary methods used in the design and construction of synthetic cells:

1. Genome Engineering

• Top-down Approach: This method involves stripping down an existing genome to its essential genes, creating a minimal genome that sustains life under specific conditions. The best-known example is the creation of the minimal bacterial genome by the team led by Craig Venter.

• Bottom-up Approach: This involves building a genome from scratch, selecting genes based on desired cellular functions. This approach allows for greater customization but requires deep understanding of gene functions and interactions.

2. Synthetic Genomics

• Whole Genome Synthesis: Using chemical synthesis to create entire chromosomes or genomes based on designed or natural sequences. This allows for the introduction of extensive modifications and the incorporation of non-natural base pairs.

• Genome Transplantation: Transferring a synthetic genome into a cellular chassis from which the native genome has been removed, effectively reprogramming the cell with the synthetic genome.

3. Protocell Construction

• Liposome-Based Models: Creating cell-like structures using liposomes to encapsulate essential cellular machinery and genetic material. These can mimic basic cell functions like metabolism and protein synthesis.

• Polymerosomes: Using polymers to form vesicles that can house biochemical processes, offering more stability and control compared to liposomes.

4. Biochemical Pathway Synthesis

• Metabolic Engineering: Redesigning existing metabolic pathways or constructing new ones to improve production of desired molecules or introduce new metabolic functions.

• Artificial Organelles: Designing and synthesizing organelle-like structures within cells to compartmentalize and optimize biochemical processes.

5. Xenobiology

• Expanding the Genetic Code: Incorporating non-natural amino acids into proteins by designing synthetic cells with an expanded genetic code, which can include more than the standard 20 amino acids.

• Alternative Nucleic Acid Structures: Designing synthetic cells with alternative nucleic acid structures (like XNA - xeno nucleic acids) that can store and transmit genetic information differently from natural DNA and RNA.

6. Computer-Aided Design (CAD) Tools

• Genome CAD: Using software tools to design genomes digitally before synthesis, enabling precise editing and optimization of genetic sequences.

• Simulation Tools: Simulating cellular processes and genome functionality to predict the behavior of synthetic cells before actual construction.

7. Microfluidics and Encapsulation Techniques

• Droplet Microfluidics: Encapsulating individual cells or cell components in droplets within microfluidic devices, allowing for high-throughput screening and optimization of cellular functions.

• Hydrogel Encapsulation: Using hydrogels to create a scaffold for cell-like structures, facilitating nutrient transport and waste removal.

Advantages in Production of DNA/RNA, Antibodies, and Proteins

Synthetic cells offer several advantages over traditional biological vectors used in the production of DNA, RNA, antibodies, and proteins:

1. Control and Predictability: Synthetic cells can be designed to have predictable and controllable behaviors, reducing the variability that often comes with natural biological systems.

2. Safety: By using minimal genomes and non-natural amino acids, synthetic cells can be engineered to be biocontained, meaning they are less likely to survive outside controlled environments. This reduces biosecurity risks.

3. Efficiency: Synthetic cells can be optimized for specific production processes, potentially increasing the yield and efficiency of protein or antibody production compared to traditional cell lines.

4. Customization: Synthetic cells can be tailored to produce novel proteins or RNA molecules that might be difficult or impossible to synthesize in natural organisms.

5. Reduced Competition: In traditional vectors, cellular resources are often divided between the organism's needs and the biotechnological task at hand. In synthetic cells, all resources can be directed towards the biotechnological application, potentially increasing production efficiency.

Current and Future Applications

Synthetic cells are currently used in research to understand fundamental biological processes by recreating them from scratch. They are also being explored for applications in drug delivery, biosensing, and the production of therapeutic molecules. Future developments could see synthetic cells playing crucial roles in environmental applications, such as bioremediation, and in the production of sustainable chemicals and fuels.

Overall, the field of synthetic cells in biotechnology is evolving rapidly, offering promising new tools and technologies for a variety of applications. The ability to design and construct life from its most basic components not only opens new industrial and medical avenues but also deepens our understanding of the essence of life itself.