9 September videos (review2 on vimeo lesson3 on vimeo) - Kate Adamala (MIT/U Minnesota)
We will discuss what is a synthetic cell, what are they good for, and how to make them. We will explore 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.
Review on the current state of progress towards making functional synthetic cell
A first successful example of synthetic minimal cell interfacing with live cell for signal processing
Please let me know if you cannot access some of those papers, I'll send out pdf's.
Express GFP in cell free system.
The kit
- enzyme mix at 1.33x
- plasmid P70-deGFP from Vincent Noireaux, same as used here: http://pubs.acs.org/doi/abs/10.1021/sb200016s
Please keep the enzyme mix frozen at all times - in -80 freezer or supplementing the dry ice until you're ready for experiment.
Experiment setup
1. Thaw the enzyme mix on ice (or in the fridge) immediately before use.
2. 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.
*We will know what's the stock c of DNA vector soon
3. Mix by gently pipetting up and down several times. Do not vortex.
4. Incubate at 29-30C for minimum of 2 hours, ideally longer - 4h.
5. Analyse GFP fluorescence immediately. The fluorescence of GFP can be analysed in the reaction mix, without purification. Use Nanodrop, or small volume cuvette fluorimeter.
Scale appropriately if needed.
Materials option 1 (recommended)
Cell free Tx/Tl system developed by Vincent Noireaux, www.noireauxlab.org
The shipment will contain the complete cell free expression system (enough for ~10 reactions) and a sample GFP vector.
If you choose this option, no additional material purchases necessary.
Please fill out the google doc (google doc link), making sure your shipping adress is correct and complete (including phone number) for cold package shipment.
Materials - option 2
Commercially available enzyme kit and expression vector from Addgene.
This might be a good option if you are worried that there might be problems with shipping a bio hazard package from academic lab in the US to your lab location.
1. vector: pIVEX-SNAP-GFP (https://www.addgene.org/46846/)
Alternatively: any vector with T7 promoter and T7 terminator, green fluorescent protein variant (or other fluorescent protein)
You will need minimum of ~100ng per student; doesn't matter which strain is used to propagate it, does not have to be endotoxin free miniprep.
2. PURExpress In Vitro Protein Synthesis Kit (https://www.neb.com/products/e6800-purexpress-invitro-protein-synthesis-kit)
Alternatively: other bacteria cell free tx/tl will work. Email me with your replacement idea.
Equipment
fluorimeter
standard lab equipment: pipetors, vortex, tubes
Design synthetic cell biosensor, reactor or actuator.
This assignment requires access to the original scientific literature. I can provide all the papers listed on the reading list below, and I will be happy to email you any other papers you find you need.
I cannot post papers in dropbox in advance, I'm pretty sure that would not be OK copyright-wise.
If you cannot access original literature, you can design your synthetic cell using commonly known bioparts, and we can later discuss the feasibility of using it in synthetic cell setting.
Guidelines below. Please use those questions as a design roadmap, and as a homework report please write up answers to those questions.
1. Pick a function.
1A What would your synthetic cell do? What is the input and what is the output.
1B Could this function be realized by cell free Tx/Tl alone, without encapsulation?
1C Could this function be realized by genetically modified natural cell?
1D Describe the desired outcome of your synthetic cell operation.
2. Design all components that would need to be part of your synthetic cell.
2A What would be the membrane made of?
2B What would you encapsulate inside? Enzymes, small molecules.
2C Which organism your tx/tl system will come from? is bactrerial OK, or do you need mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian).
2D How will your synthetic cell communicate with the environment? (hints: are substrates permeable? or do you need to express membrane channel?)
3. Experimental details
3A List all lipids and genes (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick actual gene)
3B How will you measure the function of your system?
Bonus: “A picture is worth a thousand words”. The entire homework could be done in one, detailed and annotated scheme showing all the components of your synthetic cell in desired operation.
If possible, please include references (not only original papers, websites are OK too).
Some useful reviews for getting started:
1. Stano, P. et al. Semi-synthetic minimal cells as a tool for biochemical ICT. BioSystems 109, 24–34 (2012).
2. Luisi, P. L. & Stano, P. Synthetic biology: Minimal cell mimicry. Nat. Chem. 3, 755–756 (2011).
3. Stano, P. & Luisi, P. L. Semi-synthetic minimal cells: Origin and recent developments. Curr. Opin. Biotechnol. 24, 633–638 (2013).
4. Torino, D., Martini, L. & Mansy, S. S. Piecing Together Cell-like Systems. Curr. Org. Chem. 1751–1757 (2013).
5. Elowitz, M. & Lim, W. a. Build life to understand it. Nature 468, 889–890 (2010).
6. Caschera, F. & Noireaux, V. Integration of biological parts toward the synthesis of a minimal cell. Curr. Opin. Chem. Biol. 22, 85–91 (2014).