Part A

1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell free expression is more beneficial than cell production.

Cell-free protein synthesis offers several key advantages over traditional in vivo methods:

1. Reduced biological regulation constraints: Cell-free systems bypass cellular regulatory mechanisms (like transcriptional repression, protein degradation pathways, and feedback inhibition) that can interfere with protein production in living cells.
2. Direct access to reaction components: Researchers can precisely control reaction conditions including pH, redox potential, cofactors, and substrate concentrations without cellular membrane barriers.
3. Ability to produce proteins toxic to living cells: Cell-free systems can synthesize proteins that would otherwise kill host cells or trigger stress responses that reduce yield.
4. Rapid prototyping capability: New designs can be tested in hours rather than days, accelerating design-build-test cycles for protein engineering.

Two specific beneficial applications include:

1. Wearable biosensors that incorporate freeze-dried cell-free systems activated by hydration, allowing for stable, portable diagnostic devices that can detect pathogens or environmental toxins without requiring living cells.

2. High-throughput protein screening for drug discovery, where cell-free systems enable the rapid production and testing of numerous protein variants without the time-consuming steps of cell transformation, growth, and extraction.

3. Describe the main components of a cell-free expression system and explain the role of each component.

A cell-free expression system typically contains the following essential components:

1. DNA template: Provides the genetic instructions encoding the protein of interest. This can be in the form of plasmid DNA or linear DNA fragments containing the gene with appropriate regulatory elements (promoter, ribosome binding site, terminator).
2. Transcription machinery: RNA polymerase and transcription factors that read the DNA template and synthesize mRNA. In bacterial-based systems, this typically includes T7 RNA polymerase.
3. Translation machinery: Components needed to convert mRNA into protein:
   • Ribosomes: Molecular complexes that serve as the site of protein synthesis
   • tRNAs: Transfer RNAs that deliver specific amino acids to the growing polypeptide chain
   • Aminoacyl-tRNA synthetases: Enzymes that attach specific amino acids to their corresponding tRNAs
   • Translation factors (initiation, elongation, and release factors): Proteins that facilitate the different stages of translation
4. Energy regeneration system: Provides and maintains ATP and GTP levels needed for transcription and translation:
   • Energy sources (ATP, GTP)
   • Enzymes for energy regeneration (e.g., creatine phosphate/creatine kinase or phosphoenolpyruvate/pyruvate kinase)