Week 9 Homework

Homework: Cell-Free Systems and Synthetic Minimal Cells

General homework questions

1. Advantages of cell-free protein synthesis

Cell-free protein synthesis is useful because it is faster and easier to control than expression in living cells. Since there are no cells to keep alive, I can directly change the DNA amount, salts, cofactors, energy source, and reaction conditions.

It is especially useful for making toxic proteins, because the protein does not have to be safe for the cell. It is also useful for quickly testing many genetic designs before putting them into cells.

2. Main components of a cell-free expression system

A cell-free expression system needs a DNA or mRNA template, ribosomes, tRNAs, amino acids, enzymes for transcription and translation, salts, buffer, and cofactors. It also needs an energy source, plus a way to regenerate energy, so the reaction can keep making protein.

3. Why energy regeneration is important

Energy regeneration is important because making RNA and protein uses a lot of ATP and GTP. Without a way to restore these energy molecules, the reaction would stop quickly.

One way to do this is to add an energy regeneration system such as phosphoenolpyruvate or creatine phosphate. These help keep ATP available during the reaction.

4. Prokaryotic versus eukaryotic cell-free systems

Prokaryotic systems, like E. coli extract, are usually cheaper, faster, and give high protein yield. I would use this for GFP or a bacterial enzyme, because those proteins usually do not need complicated folding or modifications.

Eukaryotic systems are better for proteins that need more complex folding or post-translational modifications. I would use a eukaryotic system for a human membrane receptor or secreted protein.

5. Optimizing a membrane protein

To optimize a membrane protein, I would test different DNA concentrations, temperatures, magnesium levels, and reaction times. I would also test liposomes, nanodiscs, or mild detergents, since membrane proteins often need a membrane-like environment.

The main problems would be low yield, aggregation, and incorrect folding. I would measure the result using fluorescence, binding, or activity assays.

6. Reasons for low protein yield

A low yield could happen because the DNA template is bad or the concentration is wrong. I would try a fresh template and test different DNA amounts.

It could also happen because the reaction conditions are not good. I would vary magnesium, potassium, temperature, and incubation time.

A third possibility is that the protein is hard to fold. In that case, I would try lower temperature, folding helpers, or a different cell-free system.

Homework question from Kate Adamala

1. Function

My synthetic minimal cell would detect theophylline and produce GFP. The input is theophylline outside the synthetic cell, and the output is GFP fluorescence inside the cell-like vesicle.

This could be done with cell-free Tx/Tl alone, but encapsulation keeps the components together and makes it more like a simple cell. It could also be done in a modified living cell, but a synthetic cell is simpler and does not grow or mutate.

The desired outcome is GFP expression only when theophylline is present.

2. Components

The membrane would be made from phospholipids and cholesterol. Inside the vesicle, I would put bacterial cell-free Tx/Tl extract, amino acids, NTPs, energy mix, salts, buffer, and DNA for GFP controlled by a theophylline riboswitch.

I would use a bacterial Tx/Tl system because this design only needs a small-molecule sensor and GFP expression. The synthetic cell would communicate with the environment by allowing theophylline to cross the membrane. If that does not work well, I would add a small pore or transporter.

3. Experimental details

Lipids: POPC and cholesterol.

Genes: GFP under a T7 promoter with a theophylline-responsive riboswitch.

Other components: bacterial Tx/Tl extract, amino acids, NTPs, energy regeneration mix, salts, and buffer.

I would measure the system by comparing fluorescence with and without theophylline. If the system works, the sample with theophylline should have much stronger GFP signal.

Homework question from Peter Nguyen

My idea is a freeze-dried cell-free paper test for water contamination.

The paper would contain dried cell-free reactions. When someone adds a water sample, the reaction rehydrates. If the target contaminant is present, the sensor turns on a visible reporter, such as a color change or fluorescence.

The societal problem is that many places need cheap and simple water testing. This could be useful because it would not require a full lab.

The main limitation is that the reaction would probably be one-time use and could lose activity during storage. I would address this by freeze-drying with stabilizers like trehalose and sealing the paper from humidity.

Homework question from Ally Huang

1. Background

A useful space biology problem is checking whether stored or recycled water has microbial contamination. In space, equipment, time, and storage are limited, so a small test would be valuable.

2. Target

I would target bacterial 16S rRNA as a general marker of bacterial contamination.

3. Relation to space biology

This target is relevant because water systems in spacecraft need to stay safe. A cell-free sensor could detect contamination without needing to grow bacteria in culture.

4. Research goal

The goal is to make a freeze-dried cell-free sensor that detects bacterial contamination in spacecraft water. This would be lightweight, easy to store, and simple to activate with a water sample.

5. Experimental plan

I would make freeze-dried cell-free reactions containing the sensor and reporter. I would test clean water as a negative control and water with added bacterial RNA or DNA as a positive control. Then I would measure color change or fluorescence after rehydration.

Final Project: Slide added, Twist and other reagants submitted.