In class, we discussed what is a synthetic cell, what are they good for, and how to make them. We saw examples of synthetic cells used to make stuff (proteins, metabolites), as well as examples of synthetic cells interfacing with biology, for readout and control of natural cells.
1. Pick a function.
- What would your synthetic cell do? What is the input and what is the output.
The synthetic minimal cell that we designed this week (see Fig.1), is meant to be a biological deodorant! The input to the system are odorants (volatile molecules that act as olfactory stimuli) of a bad smell, e.g. urea from urine in a toilet, and the output are odorants of a nice smell e.g. vanillin - the odorant of vanilla.
- Could this function be realized by cell free Tx/Tl alone, without encapsulation?
No, it could not, as olfactory receptors need a membrane to function. The papers cited on Fig. 1, show successful expression of a mammalian olfactory receptor in a liposome-like structure, using an ensemble of mammalian Tx-Tl systems.
- Could this function be realized by genetically modified natural cell?
Possibly yes, but it would limit the repertoire of possible odorants, as some of them might be toxic to the cell, or be antagonists for native receptors.
- Describe the desired outcome of your synthetic cell operation.
The artificial cell, will have a set olfactory receptors, like the ones we have in our nose that would be activated by the mal-odorants and subsequently would trigger the release of nice smelling odorants, through a gated membrane channel.
2. Design all components that would need to be part of your synthetic cell.
- What would be the membrane made of?
I will try to replicate the Small unilamellar vesicles (SUV) created on Ritz et al. paper, so the membrane will be made off dioleoylphosphatidylcholine(DOPC, 2 mM) phospholipids.
- What would you encapsulate inside? Enzymes, small molecules.
Inside I would encapsulate the enzymes required for directing the expressed receptor to inhabit functionally the SMC membrane as well as odorants like vanillin or eugenol to be released once the system gets activated by the detection of an offensive odorant.
- Which organism your tx/tl system will come from?
Quoting from the paper cited in the figure:
Furthermore, OR5 membrane topology is depended on the genetic origin of the expression system: rabbit reticulocyte lysates and bacterial lysates lead to comparable membrane insertion patterns, but with higher fractions of membrane insertion for the mammalian system. Expression of OR5 by wheat germ extract resulted in a divergent membrane insertion pattern. Expression in an insect cell extract showed no detectable membrane insertion at all.
Thus it looks promising to use the rabbit Tx-Tl system (RRL, TNT quick coupled transcription/translation, Promega). Although in our lab we have expressed olfactory receptors successfully using Vincent Noireux Tx-Tl system that was provided as part of our experimental assignment! Thus, I will use it also and compare the results.
- How will your synthetic cell communicate with the environment?
It will communicate using the olfactory receptor expressed in the membrane. Olfactory receptors are G-Protein coupled receptors (GPCRs), the largest and most diverse group of membrane receptors in eukaryotes and the core sensory apparatus of most organisms. These cell surface receptors act like an inbox for messages in the form of light energy, peptides, lipids, sugars, and proteins. GPCR's bind molecules (also called ligands) from the outer environment and trigger a secondary messenger pathway inside the cell by the activation of a G-Protein near their C-terminal inside the cell. You can find more information here.
In this particular setup, the olfactory receptor will be specific into a set of offensive odorants and when activated will trigger the release of a set of nice smelling odorants like vanillin or eugenol.
3. Experimental details.
- List all lipids and genes involved in your system.
The basic genes and lipids are the following.
- OR5 (GenBank acc. no. P70526, 314 aa, 35.5 kDa)
- G-Protein Ga15
- Phospholipid dioleoylphosphatidylcholine(DOPC, 2 mM)
- How will you measure the function of your system?
The first thing to do would be to assay the functional expression of OR5 in the artificial membrane of the synthetic minimal cell. I propose to use the "odorant response assays for a heterologously expressed olfactory receptor" by Katada et. al where you use a luciferase reporter to identify the increase in levels of cAMP or Ca+ in the cell, which happens in case of activation of the G-Protein Ga15.
If I have a functional olfactory receptor in the membrane, then measuring the function of the system can be done just by... smelling the resulting mixture ! This would mean that the activation-coupled release of the nice smelling odorants would be successful ! I can be more specific on this step as soon as I find a robust way to release the odorants with the increase of cAMP levels inside the synthetic minimal cell....
- enzyme mix at 1.33x
- plasmid P70-deGFP from Vincent Noireaux, same as used here
Protocol (Scale appropriately if needed.)
- Thaw the enzyme mix on ice (or in the fridge) immediately before use.
- For 10uL reaction: use 7.5uL of enzyme mix, x*uL DNA vector to the final concentration 5nM, add water to the final reaction volume.
- Mix by gently pipetting up and down several times. Do not vortex.
- Incubate at 29-30C for minimum of 2 hours, ideally longer - 4h.
- Analyse GFP fluorescence immediately. The fluorescence of GFP can be analysed in the reaction mix, without purification. Use Nanodrop, or small volume cuvette fluorimeter.
With total 65ul we want 75% enzyme mix and 25% plasmid+water
We know that Kate's GFP plasmid is around 5kbp, so we used this website: https://www.neb.com/tools-and-resources/usage-guidelines/nucleic-acid-data to estimate the pmol of our plasmid. Our plasmid is comparable to pBR322, which is 1ug = 0.35 pmol = 2.1 X 1011 molecules.
After our conclusions we determined that for the 10uL reaction we will use 7.5uL of enzyme mix and 1uL of Kate's GFP plasmid.
As the Tx-Tl system was already mixed and thus was highly active, we had to thaw it from -80C directly before use. It became liquid a couple of minutes after holding it tightly in a glove.
We made the mix and then we waiting for about 4 hours.
We used a plate reader as spectrophotometer to measure the GFP expression. We compared to a
control (well with
nothing inside), and there was a noticeable peak about 5 times the control.
We tried to visualize the GFP expression with blue light but were unable to see anything.