5.2 Molecular Building Blocks

Molecular Building Blocks refers to the individual, non-living chemical and biochemical parts that are combined during bottom-up synthetic cell construction to produce a cell-like system. These blocks include nucleic acids, proteins, ribosomal machinery, enzymes, membrane-forming lipids, energy-carrying molecules, metabolic substrates, cofactors, and in some cases nonbiological components, each of which must be considered in terms of its provenance, purity, and compatibility with the other parts of the system.

Nucleic Acid Components

DNA and RNA as Informational Molecules

Nucleic acid components include synthesized or purified DNA and RNA molecules that carry the genetic instructions used within a bottom-up system, encoding the genes, regulatory sequences, or templates needed for a given function.

Design Flexibility of Nucleic Acids

Because these components are typically produced through chemical synthesis, their sequence can be precisely designed in advance, allowing researchers to specify exactly which genetic elements are present without inheriting any unrelated genetic material.

Protein Components

Functional Molecules for Structure and Catalysis

Protein components are purified or recombinantly produced proteins introduced into the system to perform structural, regulatory, or catalytic roles, such as forming pores in a membrane or acting as a transcription factor.

Sourcing Considerations for Proteins

Because proteins are complex molecules that are difficult to synthesize chemically at scale, they are typically produced in living expression systems and then purified before use, introducing a point at which biological production intersects with an otherwise non-living assembly process.

Ribosomal Machinery

The Core Translation Apparatus

Ribosomal machinery refers to the ribosomes and associated translation factors included in a bottom-up system to enable the synthesis of proteins from messenger RNA templates within the constructed cell.

Necessity for Protein Synthesis Within the System

Without functional ribosomal machinery, a bottom-up system cannot produce its own proteins internally and must instead rely entirely on externally supplied, pre-made proteins, limiting the system's capacity for self-directed function.

Enzymatic Components

Catalysts for Biochemical Reactions

Enzymatic components are purified enzymes added to catalyze specific biochemical reactions within the system, such as energy regeneration reactions or steps in a defined metabolic pathway.

Precision Enabled by Defined Enzyme Sets

Because each enzyme is individually selected and added, researchers can precisely define which biochemical reactions the system is capable of performing, avoiding the presence of unrelated enzymatic activities found in natural cells.

Membrane-Forming Components

Lipids and Their Role in Compartmentalization

Membrane-forming components, primarily lipids, self-assemble into bilayer structures that enclose the other molecular building blocks, creating the compartment boundary that gives the system its cell-like character.

Variability in Lipid Composition

The specific composition of membrane-forming lipids can be varied to adjust membrane properties such as permeability, stability, and the ability to incorporate membrane proteins, making lipid selection an important design parameter.

Energy-Carrying Molecules

Powering Internal Reactions

Energy-carrying molecules, such as those that supply chemical energy for enzymatic reactions, are included to power the biochemical processes occurring within the system, since a bottom-up construct typically lacks the full biosynthetic capacity to generate its own energy carriers from scratch.

Dependence on External Supply

In many bottom-up systems, these energy-carrying molecules must be periodically replenished from outside the compartment, representing a key point of external dependency characteristic of early-stage bottom-up constructs.

Metabolic Substrates

Raw Materials for Biochemical Conversion

Metabolic substrates are the small molecules supplied as starting materials for the enzymatic or metabolic reactions the system is designed to carry out, providing the raw material that enzymes act upon.

Selection Based on Intended Function

The specific substrates included are selected based on the target function of the system, ensuring that only the necessary starting materials for the desired reactions are present, consistent with the precise component control characteristic of bottom-up construction.

Cofactor Components

Small Molecules Supporting Enzyme Function

Cofactor components are small molecules or ions required by certain enzymes to carry out their catalytic function, without which an otherwise present enzyme would remain inactive.

Importance of Matching Cofactors to Enzymes

Because each enzyme may require a specific cofactor, careful matching between included enzymes and their necessary cofactors is required to ensure that all intended reactions can actually proceed within the constructed system.

Nonbiological Component Inclusion

Synthetic Materials Beyond Biological Molecules

Nonbiological component inclusion refers to the deliberate use of synthetic materials, such as artificial polymers or engineered nanoparticles, alongside biological molecules to achieve functions that natural components cannot easily provide.

Expanding the Design Space

Including nonbiological components expands the range of possible system behaviors beyond what purely biological molecules allow, though it also requires additional testing to confirm compatibility with the surrounding biological components.

Building Block Provenance

Tracing the Origin of Each Component

Building block provenance refers to documenting the specific source of each molecular component, whether obtained through chemical synthesis, purification from a biological source, or recombinant production, since provenance affects both purity and potential for unexpected contaminants.

Relevance to Reproducibility

Clear provenance records support reproducibility, allowing other researchers to obtain or produce components through the same route and reasonably expect comparable results.

Building Block Purity

Removing Unwanted Contaminants

Building block purity refers to the degree to which each molecular component is free from unintended contaminants, such as residual host-cell proteins in a recombinantly produced protein preparation.

Consequences of Insufficient Purity

Insufficient purity can introduce unaccounted-for biochemical activity into the system, confounding interpretation of results and undermining the precise component control that bottom-up construction is intended to provide.

Building Block Compatibility

Ensuring Components Work Together

Building block compatibility refers to the requirement that molecular components function correctly under shared conditions, such as a common pH, ionic strength, and temperature, once combined within the same compartment.

Testing for Compatibility Before Assembly

Compatibility is often tested for pairs or small groups of components before full assembly, since combining components that individually function well under different optimal conditions can result in a system where none of the components perform as expected.