Week 9 Week 9 — Cell-Free Systems

General homework questions

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.

Advantages of cell-free protein synthesis (CFPS) over traditional in-vivo methods:

(i) Greater flexibility and control: Given that cells do not need to stay “alive” and the absence of a cell wall, it is possible to manipulate cells in real time; add chaperones, cofactors etc [1].

(ii) Rapid development of prototypes: Where in-vivo methods require cloning DNA into plasmids and transforming host cells, the CFPS allows us to essentially ‘drag and drop’ DNA with raw PCR products and observe protein expression in short periods of time (e.g. hours) [2]

Cases where CFPS provides benefits over in-vivo methods:

(i) Expression of toxic/dangerous antimicrobial peptides, potent neurotoxins, or complex membrane proteins in vivo. Usually the host cell would ‘die’ before reaching a large protein yield, as the CFPS is technically dead, it can synthesize toxic therapeutics and viral vectors that would be impossible to harvest from living cultures [2]

(ii) The open environment lets you easily swap natural amino acids for synthetic ones, enabling efficient, site-specific incorporation of non-standard amino acids (nsAAs) without competing with host metabolism [2]

  1. Khambhati K, Bhattacharjee G, Gohil N, Braddick D, Kulkarni V, Singh V. Exploring the potential of cell-free protein synthesis for extending the abilities of biological systems. Front Bioeng Biotechnol. 2019;7:248.
  2. Silverman AD, Kelley-Loughnane N, Jewett MC. Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet. 2020;21(3):151-70.

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

(i) Cell extract (machinery): Derived from lysed cells (like E. coli), this extract provides the core transcriptional and translational machinery, including ribosomes and RNA polymerase, required to build the protein

(ii) Genetic template (blueprint): The DNA plasmid or RNA template that contains the specific gene sequence of the target protein we want to express

(iii) Nucleotides and amino acids (building blocks): Nucleotides—Adenosine triphosphate (ATP), Guanosine triphosphate (GTP), Cytidine triphosphate (CTP), and Uridine triphosphate (UTP)—are supplied for ribonucleic acid (RNA) synthesis (transcription), while transfer RNAs (tRNAs) pair with messenger RNA (mRNA) to deliver the amino acids necessary for protein synthesis (translation)

(iv) Energy systems: immediate energy sources like adenosine triphosphate (ATP) are paired with intermediate metabolites like 3-phosphoglycerate (3-PGA) or phosphoenolpyruvate (PEP) to continuously regenerate energy and maintain reaction stability.

(v) Buffers & cofactors (Environmental conditions):

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (buffer to maintain a stable pH for optimal enzyme activity)

Mg: Magnesium (cofactor for transcription and translation enzymes)

DTT: Dithiothreitol (reducing agent that maintains a non-oxidizing environment to protect protein residues)

Sodium Oxalate: This is already the full chemical name (there is no abbreviation here, though its chemical formula is Na₂C₂O₄) (prevent magnesium precipitation, stabilizing the ionic balance)