https://pages.htgaa.org/2026a/tammy-sisodiya/homework/week-09-hw-cell-free-systems/index.htmlHomework Part A: General and Lecturer-Specific Questions 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. If we wanted to produce a toxic protein / protein deposits which are misfolded, non-functional, and often highly stable multi-molecular structures - restriction enzymes which cut and edit DNA, cytotoxic proteins (e.g. immune cell related molecules such as perforins which puncture cell membranes and granzymes which trigger cell apoptosis, both produced by NK cells), the cell free expression allows production without affecting cell viability in comparison to in vivo methods. Due to the barrier of cell membranes being removed in cell-free systems, labeled or unnatural amino acids can be put in the mix (of organelles like ribosomes) for targeted, specialised protein synthesis: for example in in vivo labeling for NMR or fluorescence studies. In in vivo methods, the cell system’s energy and resources can be divided between different organelles, whereas in CFS the energy and resources can solely be focused on making the target protein. You can do real time tracking of protein synthesis using spectrophotometry or other analytical methods in CFS due to the large, uncomplicated system comparatively to in vivo methods where the cell has many organelles. Conditions of the reaction can be controlled; variables such as temperature, redox potential and pH can be changed and finetuned without worrying about causing host death. Cloning and transformation steps are skipped, as we can use linear DNA or circular plasmid DNA directly. Two cases where CFS is beneficial over in vivo methods;Hugoen