Week 9 HW: 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.

Cell-free protein synthesis offers a number of advantages over traditional in vivo methods. An important advantage is the fact that cell-free systems are much more time efficient in comparison to their in-vivo counterparts. Cell-free systems also can operate using linear DNA fragments, whereas in-vivo systems necessitate the use of plasmid DNA.  

Genetic expression in cell-free systems is directly proportional to the concentration of DNA that is present, this allows for highly specific protein production quantities. 


Cases where cell-free expression is more beneficial than cell production:

  1. Cell-free systems allow for the production of proteins that would otherwise be toxic to cellular systems. 

  2. Cell-free systems could potentially result in higher throughput for biofuel production as compared to in-vivo mechanisms. 


Nico J Claassens, Simon Burgener, Bastian Vögeli, Tobias J Erb, Arren Bar-Even,

A critical comparison of cellular and cell-free bioproduction systems,Current Opinion in Biotechnology, Volume 60, 2019, Pages 221-229, ISSN 0958-1669, https://doi.org/10.1016/j.copbio.2019.05.003. (https://www.sciencedirect.com/science/article/pii/S0958166918301861)

Rollin, J.A., Tam T. K. and Zhang Y.-H. P. , (2014) ‘New biotechnology paradigm: cell-free biosystems for biomanufacturing’, Green Chemistry, 16(9), pp. 3248–3255.

https://pubs.rsc.org/en/content/articlelanding/2013/gc/c3gc40625c#!divCitation

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

The core components of the cell-free expression system include cell-free extracts, template DNA, and energy sources (such as ATP, GTP, etc.). 

Cell extracts form the basis of the in vitro expression system, containing all the necessary cellular machinery, such as ribosomes, transcription factors, and modifying enzymes. Template DNA encodes the target protein and can be obtained through PCR amplification or synthesis.

  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 provision regeneration is critical in cell-free systems, as there is no cellular metabolic function to continuously provide the necessary energy for any cellular expression.  

A method to ensure continuous ATP supply in cell-free experiments is the addition 

A common strategy for achieving phosphorylation of ADP is using pairs of compounds with high-energy phosphate bonds, along with their cognate kinases such as acetylpjosphate/acetate kinase, phospoenolpyruvate/pyruvate kinase or creatine phosphate/xreatine kinase. However, use of these compounds inevitably results in accumulation of inorganic phosphate in the reaction mixture. 

Lee, Kyung-Ho & Kim, Dong-Myung. (2018). Recent advances in development of cell-free protein synthesis systems for fast and efficient production of recombinant proteins. FEMS microbiology letters. 365. 10.1093/femsle/fny174. 


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

Prokaryotic cell-free expression systems often based on E. coli extract, have a high protein yield, simple cultivation and cell growth and lysate preparation. They’re cost efficient and easy to genetically engineer. The disadvantages of these systems, however, are that they have limited posttranslational modifications, no endogenous membrane structures for the synthesis of integral membrane proteins and only native prokaryotic chaperones are available. It might not be able to fold eukaryotic proteins correctly.  (Zemella, 2015)

Eukaryotic cell-free systems offer a wider variety in extract sources. The main disadvantages that often arise with these cell free systems are high cultivation costs and low protein yield in mammalian and yeast cells. Plant-based extracts like wheat germ and tobacco BY-2 do seem to have a higher protein yield, but relative to prokaryotic systems it is still low. However, eukaryotic CFSs have the ability to handle complex post-translational modifications. (Zemella, 2015)

In  prokaryotic cell-free expression systems I would choose to produce a protein that incorporates non-canonical amino acids, as these systems make this feasible. A protein I would produce in a eukaryotic cell-free expression system would be GFP.    

Zemella, A., Thoring, L., Hoffmeister, C., & Kubick, S. (2015). Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. Chembiochem : a European journal of chemical biology, 16(17), 2420–2431. https://doi.org/10.1002/cbic.201500340

  1. 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.

Cell-free expression of membrane proteins struggles with hydrophobic aggregation and ribosome stalling during nascent chain synthesis.

Setup: Use PURExpress or E. coli lysate with a GFP-fused bacterial membrane protein. Add liposomes (PC:PE mix) or nanodiscs (0.1-1 μM) at t=0. Test lipid variants and N-terminal modifications in 20 μL reactions at 25°C for 4h.

Readouts: Total GFP fluorescence; centrifugation for soluble/insoluble fractions; protease protection for insertion.

Solutions: Nanodiscs promote co-translational insertion; optimized lipids aid folding. Select condition maximizing functional, membrane-associated yield. Controls: soluble GFP, no-lipid baseline. Boosts often 5-10x. 

  • Roos C, Kai L, Haberstock S, Proverbio D, Ghoshdastider U, Ma Y, Filipek S, Wang X, Dötsch V, Bernhard F. High-level cell-free production of membrane proteins with nanodiscs. Methods Mol Biol. 2014;1118:109-30. doi: 10.1007/978-1-62703-782-2_7. PMID: 24395412.

  1. 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.

Three possible reasons for a low yield in a cell-free system:

  1. Chosen prokaryotic or eukaryotic extract is not suitable for high yield protein output, a troubleshooting strategy for this would be to shift to a prokaryotic extract to then compare the resulting protein yield.  

  2. Initial DNA concentration and necessary components are too low, resulting in low output, alter the concentrations of components within the set-up to maximise ratios for highest protein yield. 

  3. The chosen experimental set-up is a batch system, resulting in lower protein yield.  If possible, switch toa CECF reaction system. (Schwarz, 2007)


  • Schwarz, D. et al. Preparative scale expression of membrane proteins in E.coli based continuous exchange cell-free systems. Nat. Protocols 2, 2945-57 (2007)


Homework question from Kate Adamala

Design an example of a useful synthetic minimal cell as follows:

  1. Pick a function and describe it.

    1. What would your synthetic cell do? What is the input and what is the output?

In line with my final project research, my proposed synthetic cell would hold the capacity to degrade PFAS particles, based on the discovery of enzymatic degradation of PFAS by soil bacteria found in Portugal. The input therefore would be various compounds within the category of PFAS particles. The output would be chain-shortened, non-toxic molecules.


  1. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

It would be very interesting to have this function be realized by cell-free Tx/Tl alone, as the enzymatic degradation system would be deployable much faster and much safer than an in-vivo counterpart. 


  1. Could this function be realized by genetically modified natural cell?

This function could be realized by genetically modified natural cells, as the envisioned functionality was first observed in L. portucalensis F11. However, this strain of bacteria takes a significantly long time to metabolise these compounds. Transplanting this enzymatic function into a different chassis like E.coli K12 would also come with its own challenges, as efflux systems would have to be neutralised due to the toxic nature of the PFAS particles. 

  1. Describe the desired outcome of your synthetic cell operation.

The desired outcome of my synthetic cell operation would be the degradation of PFAS in its surrounding 

  1. Design all components that would need to be part of your synthetic cell.

    1. What would be the membrane made of?

The membrane would be a zwitterionic phospholipid membrane consisting of DOPC and DOPG. 


Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Structural-comparison-of-DOPG-and-DOPC-lipid-headgroups-DOPG-A-headgroup-is-composed-of_fig1_340707406 [accessed 6 Apr 2026]

  1. What would you encapsulate inside? Enzymes, small molecules.

Inside of the membrane I would encapsulate the Tx/Tl system for the PFAS degrading enzyme, as well as all necessary resources for polypeptide creation. 

  1. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian)

The organism my Tx/Tl system would come from would be bacterial, as described prior in this exercise. 

  1. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

The PFAS particle substrates are permeable, therefore there would not be a need for additional membrane channels. 


Sangwoo Ryu, Emi Yamaguchi, Seyed Mohamad Sadegh Modaresi, Juliana Agudelo, Chester Costales, Mark A. West, Fabian Fischer, Angela L. Slitt, Evaluation of 14 PFAS for permeability and organic anion transporter interactions: Implications for renal clearance in humans, Chemosphere, Volume 361, 2024, 142390, ISSN 0045-6535,

https://doi.org/10.1016/j.chemosphere.2024.142390.


  1. Experimental details

    1. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)

Lipids: DOPC and DOPG

Genes: Yet to be fully documented sequences for the enzymatic degradation of PFAS particles. 


  1. How will you measure the function of your system?

I would utilize mass spectrometry to measure the degradation of the initial molecules. 


Homework question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field — Architecture, Textiles/Fashion, or Robotics — and propose an application using cell-free systems that are functionally integrated into the material. Answer each of these key questions for your proposal pitch:

  • Write a one-sentence summary pitch sentence describing your concept.

As described in my answers to Kate Amala’s homework questions, I would propose a cell-free system that is able to degrade PFAS particles, which can be adapted to function as a freeze-dried cell-free system. 

  • How will the idea work, in more detail? Write 3-4 sentences or more.

PFAS pollution is one of the great pollutive issues we face today. Recent scientific evidence shows the potential of certain bacteria to biodegrade these particles. By utilizing lyophilization technology, cell-free versions of this enzymatic system can be distributed world wide. Freeze-dried systems are activated by water; this component can be utilised to our advantage when the system is deployed in PFAS contaminated waters.  

  • What societal challenge or market need will this address?

This will address the need to remediate against the presence of PFAS particles in our environment. 

  • How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

While cell-free reactions depend on water to activate, this can be used to our advantage when dealing with contaminated waters. While one-time use has its downsides, it could prove to be advantageous when deploying the system into the environment. We cannot predict what uncontrolled accumulation of these components would do in the environment in the long run, therefore their temporary nature would be less hazardous. 

Homework question from Ally Huang

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space!

For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/ .


  1. Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words)


It is well-known that exposure to cosmic radiation is a great problem in space exploration. An important possible health risk is the development of cancer. However, cancer diagnostics are 

more difficult due to environmental and spatial factors in space. 


Carcinoembryonic antigen (CEA) is a nonspecific serum biomarker that is elevated in many malignancies, including colorectal cancer, medullary thyroid cancer, breast cancer, and mucinous ovarian cancer. The ELISA method detects antigen-antibody interactions by using enzyme-labelled conjugates and enzyme substrates that generate colour changes; it is often used to assess CEA-values in blood. 


Outside of cancer diagnostics, the availability of antibodies is vital for various fields of research. 


  1. Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words)

The main molecular target I propose to study is the anti-CEA antibody, which is a receptor protein used in ELISA detection of CEA concentration in blood. 


  1. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

Currently, production of antibodies is predominantly done through Polyclonal Antibody Production, a process that often involves significant animal cruelty, is relatively inefficient and can often take months.  Besides a matter of optimisation, moving towards a cell-free synthesizing methodology is an important matter of animal welfare. 


While animal rights are still incredibly important in a space setting, it is obvious to state that this technology would not be available in the first place. Besides moral and ethical objections, taking animals into space for this purpose would additionally come with significant additional challenges in terms of resources, time and spatial capacities. 


Therefore ensuring that cell-free synthesis of antibodies is functional in space is vital in the development of sustainable space exploration. 


  1. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

Goal:

The research goal of this project is understanding the possibilities of synthesizing anti-CEA antibodies in outer space for disease diagnostics, which would be indicative of the synthesizing possibilities of other antibodies. 


Reasoning:

As stated prior, the synthesis of antibodies in space is an important technology to hone in the development of space adapted diagnostics. Anti-CEA antibodies are selected for this experiment as their CEA counterpart is a key indicator for various cancers in the body. 

Moreover, the BioBits® cell-free protein expression system could be a suitable candidate for this experiment as its lyophilised system would be applicable for experiment reproduction in space. 


  1. Outline your experimental plan - identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words)

The samples I would test in my experiment would be synthetic genetic pathways for protein production, specifically for anti-CEA antibodies. These pathways would be developed based on sequencing data and antibody synthesis precedents obtained from literature review. 

The DNA would be obtained from in-vivo specimens and amplified using PCR. 

Type of data measured: In line with the BioBits® cell-free protein expression’s functionality, the measured data would be a GFP readout that would be in proportion to the expression of the antibodies.