10.13 Synthetic Cell Translation System Engineering

Synthetic Cell Translation System Engineering refers to the deliberate modification of translation machinery within a synthetic cell system to expand its capabilities beyond standard protein synthesis, enabling functions such as the incorporation of novel building blocks or the creation of translation pathways that operate independently of the system's native machinery. This engineering spans codon usage optimization, supplementation of transfer RNA supply, ribosome engineering, use of orthogonal ribosomes and transfer RNA pairs, engineering of aminoacyl-tRNA synthetases, incorporation of noncanonical amino acids, reassignment of stop and sense codons, modification of release factors, control of mistranslation, and verification of engineered translation orthogonality.

Synthetic Cell Codon Usage Optimization

Adjusting Codon Choice to Match Available Translation Resources

Synthetic cell codon usage optimization involves selecting which synonymous codons to use when encoding a given protein sequence, choosing codons that correspond to more abundant transfer RNA species within the specific system being used.

Reducing Translation Delays Caused by Resource Mismatch

This optimization reduces the likelihood of rare codon delay, since aligning codon choice with the actual transfer RNA supply available in a given system can meaningfully improve translation speed and consistency.

Synthetic Cell Transfer RNA Supplementation

Adding Extra Transfer RNA to Address Supply Shortages

Synthetic cell transfer RNA supplementation involves deliberately adding extra quantities of specific transfer RNA species to the system, directly addressing shortages that would otherwise limit translation of transcripts using the corresponding codons.

A Direct Countermeasure to Transfer RNA Competition

This supplementation serves as a direct countermeasure to transfer RNA competition, allowing a system to support efficient translation of transcripts containing codons that would otherwise be limited by insufficient native transfer RNA supply.

Synthetic Cell Ribosome Engineering

Modifying the Ribosome Itself for Altered or Expanded Function

Synthetic cell ribosome engineering involves deliberately modifying the structure or composition of ribosomes used within the system, altering their function beyond that of an unmodified, naturally occurring ribosome.

Enabling Specialized Translation Behavior

This engineering can enable specialized translation behavior not achievable with standard ribosomes, supporting applications such as expanded genetic code use or altered fidelity characteristics tailored to a specific experimental goal.

Synthetic Cell Orthogonal Ribosome Use

Ribosomes Dedicated to a Specific, Separate Translation Task

Synthetic cell orthogonal ribosome use refers to the deployment of a specially engineered ribosome population that translates only a specific subset of transcripts, operating largely independently of the system's native ribosome population and its associated translation activity.

Value for Isolating Specialized Translation From Native Function

This orthogonal use allows specialized translation tasks, such as noncanonical amino acid incorporation, to proceed without interfering with or being interfered with by the ongoing native translation activity occurring elsewhere in the system.

Synthetic Cell Orthogonal Transfer RNA Pair

A Transfer RNA and Its Matching Enzyme Operating Independently

Synthetic cell orthogonal transfer RNA pair refers to a specifically engineered transfer RNA molecule paired with its own dedicated aminoacyl-tRNA synthetase, designed to function independently of the native transfer RNA and synthetase pairs already present in the system.

Foundation for Introducing Novel Amino Acids

This orthogonal pair provides the foundational tool needed to introduce a novel amino acid into the genetic code, since the pair can be charged with a chosen amino acid without interference from or interference with the native transfer RNA and synthetase pairs already active in the system.

Synthetic Cell Aminoacyl-tRNA Synthetase Engineering

Modifying the Enzyme That Charges a Transfer RNA

Synthetic cell aminoacyl-tRNA synthetase engineering involves modifying the specificity of an aminoacyl-tRNA synthetase enzyme so that it recognizes and attaches a novel or altered amino acid rather than one of the twenty standard amino acids it would ordinarily charge.

A Necessary Companion to Orthogonal Transfer RNA Engineering

This engineering typically proceeds hand in hand with the design of an orthogonal transfer RNA, since a functional orthogonal pair requires both a properly engineered transfer RNA and a correspondingly engineered synthetase capable of charging it with the intended novel amino acid.

Synthetic Cell Noncanonical Amino Acid Incorporation

Introducing Amino Acids Beyond the Standard Twenty

Synthetic cell noncanonical amino acid incorporation refers to the deliberate insertion of an amino acid not among the twenty standard amino acids into a growing protein chain at a specifically designated position within the sequence.

Expanding the Functional Range of Producible Proteins

This incorporation expands the functional range of proteins that the system can produce, enabling properties or chemical functionalities not achievable using only the standard set of naturally occurring amino acids.

Synthetic Cell Stop Codon Reassignment

Repurposing a Stop Signal to Encode an Amino Acid Instead

Synthetic cell stop codon reassignment involves repurposing one of the standard stop codons so that, instead of terminating translation, it instead directs incorporation of a specific noncanonical amino acid at that position.

A Common Strategy for Enabling Noncanonical Amino Acid Incorporation

This reassignment represents a commonly used strategy for enabling noncanonical amino acid incorporation, since repurposing an existing stop codon avoids the need to eliminate and reassign a codon that would otherwise encode a standard amino acid.

Synthetic Cell Sense Codon Reassignment

Repurposing a Codon That Normally Encodes a Standard Amino Acid

Synthetic cell sense codon reassignment involves repurposing a codon that would ordinarily encode one of the twenty standard amino acids, redirecting it instead toward incorporation of a noncanonical amino acid.

Greater Complexity Compared to Stop Codon Reassignment

This reassignment is generally more complex than stop codon reassignment, since it requires eliminating the codon's original use throughout the genome or template set to avoid disrupting the translation of proteins that still require the standard amino acid at that codon.

Synthetic Cell Release Factor Modification

Adjusting Release Factor Behavior to Support Codon Reassignment

Synthetic cell release factor modification involves altering the properties or availability of release factors, particularly those recognizing a reassigned stop codon, to prevent premature termination at positions where a noncanonical amino acid is meant to be incorporated instead.

Necessity for Successful Stop Codon Reassignment

This modification is often necessary to support successful stop codon reassignment, since competition between the native release factor and the orthogonal translation machinery at the reassigned codon can otherwise reduce the efficiency of noncanonical amino acid incorporation.

Synthetic Cell Mistranslation Control

Minimizing Errors Introduced by Translation System Modifications

Synthetic cell mistranslation control refers to measures taken to minimize unintended translation errors that can arise from the modifications described above, such as unintended incorporation of a standard amino acid at a reassigned codon or incorporation of the noncanonical amino acid at an unintended location.

Necessity for Reliable, Predictable Engineered Translation

This control is necessary for ensuring that engineered translation behaves predictably, since uncontrolled mistranslation would undermine confidence that a given engineered protein product actually contains the intended noncanonical amino acid at precisely the intended position and nowhere else.

Synthetic Cell Translation Orthogonality Verification

Confirming That Engineered Components Function Independently

Synthetic cell translation orthogonality verification confirms that an engineered ribosome or transfer RNA-synthetase pair actually functions independently of the native translation machinery, without unintended crosstalk that would compromise either native or engineered translation activity.

A Necessary Check Before Relying on Engineered Translation Components

This verification serves as a necessary check before relying on engineered translation components for further experimental work, ensuring that the intended separation between native and orthogonal translation activity has genuinely been achieved rather than merely assumed.