Week 9 HW: Cell-Free Systems
General homework questions
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 enable rapid protein production without the need to wait for traditional transformation and growth. This fast prototyping enable fast test of multiple genes at the same time, genetic parts, variants, and other kind of libraries. Also, other variables, such as temperature, concentration of analytes, non-canonical AAs, etc, could be tested easily in CFE. One case could be when characterizing multiple parts in an organism, where making it in CFES would be much more time efficient. Other case could be when making proteins with non-canonical AAs, making it more easy in the CFES.
Describe the main components of a cell-free expression system and explain the role of each component.
Lysate (provide cellular machinary), phosphates (e.g potassium phosphate monobasic and potassium phosphate dibasic, wich maintains a constant pH, and lowers the phosphatase activity of the lysate), AAs (for efficient protein production), ribonucleosides (nucleotide and energy metabolite precursors), magnesium glutamate. potassium glutamate (both for ions), PEG 8000 (for molecular crwowding) and the genetic component to incorporate.
Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.
For short-term the best could be the incorporation of phosphoenolpyruvate (PEP) and pyruvate Kinase (PK), wich transfer one phosphate from PEP to ADP. For medium term could be adding the enzymes for the glycolitic pathway, turning glucose-1-phosphate into pyruvate, generating ATP through glycolysis. For long term, the option of the use of photosynthetic and CO2 fixation pathways, or analogous for autonomous energy production to maintain large productions of proteins, would be crucial.
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
Prokaryotic CFES produce high yield of proteins, but doesn`t worf for folding comple eukaryotic proteins and for PTM. Eukaryotic CFES, in the other hand, enable this, but have lower yields, and higher costs. In prokaryotics CFES i could produce T5 exonuclease, and in eukaryotic CFES, monoclonal antibodys, wich need to have PTMs.
How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.
One option could be the use of lipid vesicles plus the machinary to drive the protein there, but this may require a lot of genes, wich could make all more difficult. Or maybe not. maybe 3-4 proteins that 1. bind to the liposome membrane, drive the ribosome or protein there, and enable integration of the protein there. Other option could be the use of equivalent proteins that are engineer to be soluble in water, while maintaining the same functionalites that the hydrophobic counterpart. One idea also could be the use biomolecular condensates that have an hydrphobic interior and could help fold the protein there.
Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.
Poor DNA/mRNA quantity and quality, and a troubleshoot could be checking the purity in the spectrophotometer, run agarose gel, and making enzymes digestions. Inapropiate folding, could be treated by reducing the incubation temperature, making the folding more slow. Insolubility is another problem that should be checked maybe computationally, creating soluble versions. And rapid depletion of energy substrates. For this, switching from a bacth mode to an continuous exchange cell free dialysis system could make it work more time.