Week 9 HW: Cell Free Systems

Part A: General and Lecturer-Specific Questions

General Questions

Q1. 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 two main advantages over in vivo methods: direct control and speed. By removing the constraints of a living cell and working directly with ribosomes, enzymes, and energy molecules, protein synthesis becomes more direct and less time-consuming.

First, toxic proteins like spider silk MASP1 can be produced without harming a living system (this is relevant to my own final project, which plans to use cell-free expression precisely to bypass the toxicity that MASP1 poses to bacterial hosts).

Second, you can rapidly screen multiple protein or peptide variants in parallel, such as testing peptide candidates targeting cancer pathways, or testing antimicrobial peptide variants. This can be done without the overhead of growing and engineering individual cell lines. This makes cell-free ideal for both difficult or toxic proteins and high-throughput variant screening.

Q2. Describe the main components of a cell-free expression system and explain the role of each component.

A cell-free system needs five main components. The DNA or mRNA template gives the instructions (like my MASP1 spider silk sequence from UniProt for FP).

Ribosomes read the template and build the protein. Transfer RNAs bring amino acids to the ribosome. The amino acids are the actual building blocks. An energy system (ATP) powers the whole process. You also need the right salts and pH to keep everything working. Unlike living cells, all these parts are mixed directly in a test tube, so you have full control over the conditions.

Q3. 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 critical in cell-free systems because protein synthesis requires continuous ATP. Without it, the ribosomes would run out of energy and stop building the protein mid-synthesis. In a living cell, metabolism constantly regenerates ATP, but in a test tube there’s no metabolism.

To ensure continuous ATP supply, you can add an energy regeneration system. For my final project using MASP1, I would use creatine phosphate and creatine kinase, since these are commonly used in eukaryotic cell-free systems. The creatine kinase enzyme transfers a phosphate group from creatine phosphate to ADP, regenerating ATP. If I were using a bacterial cell-free system instead, I would use PEP and pyruvate kinase, which serves the same purpose but aligns better with bacterial metabolism.

Q4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

Prokaryotic cell-free systems (like E. coli extract) are faster, cheaper, and simpler. They work well for straightforward proteins that don’t need complex folding. Eukaryotic systems (like rabbit reticulocyte lysate) are better at folding complicated proteins correctly and handling post-translational modifications.

For my final project, if I was testing the tremella fusiformis protein I would produce it in a prokaryotic E. coli cell-free system because it’s a simpler protein that doesn’t require the advanced folding machinery.

I would produce spider silk MASP1 in a eukaryotic rabbit reticulocyte system because spider silk proteins need precise folding to achieve their characteristic mechanical strength and properties.

Q5. 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.

Snow Fungus, membrane protein. Challenges: The hydrophobicity and aggregation and a way to address that is to optimize the sequence to reduce those hydrophobic regions or to add tags that help with solubility.

Q6. 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 low yield and troubleshooting strategies: (Have thought about these for FP)

Reason 1: Construct failure. Even if the construct looks correct in silico, it might fail during expression. Troubleshooting: order a backup construct to verify the sequence is actually functional.

Reason 2: Protein structure collapse. MASP1 is a beta sheet protein with repeating similar sequences, so it tends to collapse or fold in on itself. Troubleshooting: codon optimize the sequence fewer times (e.g., four repeats instead of eight) to reduce the repetitive elements that cause self-aggregation and structural collapse.

Reason 3: Energy system failure. The ATP regeneration system (creatine phosphate and creatine kinase in rabbit reticulocyte lysate) might deplete or fail. Troubleshooting: prepare a backup of the full fresh rabbit reticulocyte lysate system to ensure continuous energy supply.

Homework Question from Kate Adamala: Design a Synthetic Minimal Cell

Design an example of a useful synthetic minimal cell.

1. Function: Lyme Disease Biosensor

My synthetic cell detects Borrelia burgdorferi protein and produces a fluorescent signal as output. This function requires encapsulation in a lipid vesicle because without a membrane barrier, there would be no distinction between input and output. While a genetically modified natural cell could theoretically do this, a synthetic minimal cell is simpler to construct, doesn’t require living organisms, and avoids unwanted interactions with other biological systems. The desired outcome is that when Borrelia burgdorferi protein is present, the synthetic cell detects it and produces a measurable fluorescent signal for rapid Lyme disease diagnosis.

2. Components

The membrane would be made of biocompatible lipids (POPC and cholesterol) to avoid triggering an immune response. Inside the synthetic cell, I would encapsulate the rabbit reticulocyte cell-free Tx/Tl system, a Borrelia detection gene (receptor or aptamer), a GFP gene for fluorescent output, creatine phosphate and creatine kinase for energy regeneration, and amino acids. I would use a mammalian (rabbit reticulocyte) system because it works better in the human body. The membrane is permeable to Borrelia protein so it can enter and be detected, and GFP fluorescence is visible from outside.

3. Experimental Details

Lipids: POPC, cholesterol. Genes: Borrelia receptor/aptamer gene, GFP gene. Enzymes: rabbit reticulocyte lysate, creatine kinase. Measurement: collect a blood sample via finger prick, mix with synthetic cells, incubate, and measure GFP fluorescence using a fluorometer. Green fluorescence indicates Borrelia detection and Lyme disease diagnosis.

Homework Question from Peter Nguyen: Cell-Free Systems in Materials

Freeze-dried cell-free systems can be incorporated into materials as biological sensors or inducible enzymes. Choose one field: Architecture, Textiles/Fashion, or Robotics, and propose an application.

Field chosen: [Architecture / Textiles/Fashion / Robotics]

One-sentence pitch:

How will it work? (3-4 sentences)

What societal challenge or market need does this address?

How do you envision addressing the limitations of cell-free reactions (activation with water, stability, one-time use)?

Homework Question from Ally Huang: Mock Genes in Space Proposal

Your proposal must incorporate the BioBits® cell-free protein expression system. You may also use the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer.

Q1. Provide background on the space biology question or challenge you propose to address. Why is it significant, relevant for space exploration, and scientifically interesting? (max 100 words)

Q2. Name the molecular or genetic target you propose to study. (max 30 words)

Q3. Describe how your molecular or genetic target relates to the space biology challenge your proposal addresses. (max 100 words)

Q4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (max 150 words)

Q5. Outline your experimental plan: identify the samples you will test, necessary controls, and the type of data or measurements that will be collected. (max 100 words)

Part B: Individual Final Project

[Y ] Put your chosen final project slide in the appropriate slide deck (following the instructions on slide 1)
[Y ] Submit the Final Project selection form (if not already done)
[Y ] Begin planning your final project documentation (see guidelines)
Prepare your first DNA order and add it to the Twist ordering spreadsheet

Aim 1:

Notes / planning: