Part A - Related 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.

In cell-free protein synthesis is a new method that has been shown to give more flexibility at the time of protein production and a greater control over experimeintal variables than traditional in vivo methods. Cell-free systems do not require actual cells to carry out the reactions and make the products the researcher is interested in making. It consists of using the cellular machinery and individual components to make a molecule or product of choice. It gives the researcher more control over the variables since they chose which components need to be a part of the system. The researcher decides which cellular components are added, as well as the DNA that will be expressed. Additionally, there is more flexibility in terms of maintaining and monitoring the system; it doesn’t need the amount of monitoring an in vivo system needs, as well as less machinery.

One case in which it would be beneficial to use a cell free system would be when the product of interest is toxic to the cell. If it were to be produced in an in vivo cell, it would kill it before a significant amount of product will be made. Another case in which it would be beneficial would be when there needs to be a more efficient production and isolation of the protein. When they are produced in in vivo systems, the cells also produce their own proteins and compounds that they need to live. These products can interfere with the isolation of the protein.

https://dnadots.minipcr.com/wp-content/uploads/2019/09/DNAdots-Cell-Free-Tech-final_qnoa.pdf

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

Cellular machinery

• RNA Polymerase
  • This enzyme is responsible for translating the gene encoded in the DNA molecule into a mRNA molecule. It uses the nucleotides available to build onto the 3’OH end of a primer bonded to the begining of the sequence, forming the mRNA molecule.
• Ribosome
  • This ribozyme is composed of ribosomal RNA and proteins. It is responsible for creating a polypeptide chain from the mRNA translated from the genetic material.
• tRNA
  • This type of RNA is a helper molecule in the process of building a polypeptide. It is responsible for taking the amino acid that corresponds to the codon that is within the ribosome’s active site. It delivers the appropriate amino acid and the ribosome is responsible for adding it to the growing peptide chain.

Supplements

• Nucleotides

  • The nucleotides can be chemically synthezised. They are then added to the mix so that they are available for the polymerase to use and build the mRNA. The nucleotides in the mix should be A, U, G, and C.

• Amino acids

  • The amino acids can be chemically synthezised. They are added in order for the tRNA and the ribosome to be able to build the peptidic chain that forms the protein of choice. The 20 naturally ocurring amino acids need to be added, their ratio should depend on the protein the system is building.

• Energy source

  • Enzymes require energy to build molecules like mRNA or peptide chains. Usually the energy source is ATP, which can also be chemically synthezised.

• DNA template

  • The DNA template contains the gene or genes that code for the protein that the cell-free system will produce. It is important for the DNA sequence to have the sequences that allow for the RNA polymerase to identify it and bind to it effectively, as well as the stop codon that will signal the end of the sequence.

  https://dnadots.minipcr.com/wp-content/uploads/2019/09/DNAdots-Cell-Free-Tech-final_qnoa.pdf

3. 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.

The expression of membrane proteins is a type of production that has been especially challenging with traditional methods of production. Membrane proteins have unique characteristics that make it so they are difficult to produce outside the cell. They are amphipathic molecules, meaing they have hydrophobic and hydophilic domains.

4. 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.