Homework 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.

Example 1: The development of cell-free biosensors to detect two broad classes of analytes on the International Space Station Example 2: Production of antibody fragments (such as nanobodies or scFvs) for rapid disease testing

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

-DNA: Encodes the information to build proteins -Amino Acids: The monomers that make up functional proteins -Machinery: The transcriptional and translational machinery of the lysate -Energy: Molecules that power the reaction synthesis reactions

1. 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.

Energy regeneration is an important factor to consider because it is necessary to constantly add it to the system. This is because the absence of living cells makes it difficult to maintain energy for biochemical reactions. This can affect the performance of protein synthesis.

One method to overcome this difficulty could be testing different energy sources, as was done in a study conducted by researchers from the Department of Chemical Engineering and Applied Chemistry at Chungnam National University, Daejeon 34134, Korea.

In this study, they tested the following compounds: creatine phosphate (CP), fructose-1,6-bisphosphate (FBP), glucose-6-phosphate (G6P), and phosphoenolpyruvate (PEP), evaluating which of them resulted in the highest solubility and yield of specific recombinant proteins.

The researchers found that the use of creatine phosphate (CP) led to a higher production of target proteins, both in terms of quantity and solubility. This finding contributes to maintaining the pH of the reaction mixture throughout the entire process.

Effects of ATP regeneration systems on the yields and.pdf

1. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

-In eukaryotic cell-free expression systems, the production of antigens for antibody generation is very convenient, as no additional folding step is required.