Week 8: Cell-free Protein-folding & Synthetic Minimal Cells

Class protocol reference

Making a reaction mix of cellular components to create a cell-free protein-folding system

We made two protein transcription/translation reactions, one for eGFP (enhanced GFP), another for the FlAsH peptide (a domain, or small part, of a protein to which the fluorescent FlAsH ligand binds).

Calculating reagent volumes for a 20 ul reaction:
• General reagents: Simply remember that (initial volume)(initial concentration) = (final volume)(final concentration), where initial volume is the volume needed of the reagent stock.
• Plasmids: Given the desired concentration of plasmid in the final reaction in units of molarity, and the stock concentration of plasmid in units of nanograms/microliter (ng/ul), to determine how much volume we need to add from stock we also need to know the size of the plasmid. With that knowledge we can determine how many moles of plasmid are in one microliter of the plasmid stock solution (I use the NEBioCalculator). Moles/microliter = molarity (where fmol/ul = nM).
• General tips: Depending on the limitations of the pipettor, you may want to dilute the stock solution further so that you can use larger volumes (this will naturally result in less water needed to be added to the reaction to get it up to 20 ul). If you're doing multiple reactions, you can also prepare a "master mix" containing a multiple of all the shared reagents (you want to use more than the exact amount necessary in the master mix) - for example the GFP and FlAsH reactions only differ by the plasmid and the two FlAsH-specific reagents.

Predicting fluorescence

We can do some calculations to predict the quantum yield of the proteins that our cell-free systems are making.

Some numbers (I used the FPBase reference from above for eGFP and this paper from the Tsien lab for FlAsH peptide, and BioNumbers for transcription/translation numbers):
• Peptide length: eGFP - 238 amino acids, FlAsH - 17 amino acids
• Average E. coli transcription rate: 45 nucleotides per second (15 amino acids per second)
• Average E. coli translation rate: 15 amino acids per second
• Quantum yield (ratio of photons emitted to photons absorbed, or the likelihood that, once excited by a photon, the protein (state) will emit a photon): eGFP - 0.6, FlAsH - 0.49

We can estimate each protein's accumulated fluorescence (photons emitted) per second (assuming that the number of photons absorbed increases linearly with molecules of proteins synthesized) by adding together the transcription and translation rates, dividing by the length of the peptide, and multiplying by the quantum yield. With the numbers that I found, I estimated that FlAsH fluorescence/second is 0.86 and eGFP is 0.076, giving a FlAsH:eFGP ratio of about 11. This suggests that we would see fluorescence from the FlAsH reaction first. I found some different quantum yield numbers for eGFP, though, so...*shrugs*.

Measuring fluorescence

The maximum excitation/emission wavelengths for eGFP (the same wavelength settings can be used for FlAsH) are 488/507, according to the FPBase entry.

Fluorescence readings were taken after 12 hours of incubation at 37C.

We had two biological replicates for each protein in our group (made the exact same way, divided into two different 96-plate wells). As a set of technical replicates, another group did four biological replicates of each protein. I normalized the fluorescence reads by subtracting the average measurement from 4 blank wells. For both groups, the FlAsH replicates were much more consistent than the eGFP (average fluorescence 92380 +/- 2222 vs 38108 +/- 30946 for my group, 47133 +/- 2552 vs 39364 +/- 12117 for the other group). In both cases, FlAsH did have higher fluorescence than eGFP. Interestingly, eGFP fluorescence increased according to the order in which the samples were added to the well.

Designing a SMC

I propose to use SMCs as mechanism for delivering CRISPR-Cas systems to adult plants to perform genome engineering via a foliar spray or other liquid application.

Function

• Not really an input or output, but the SMC would be delivering a self-replenishing CRISPR-Cas system to plants, targeting the gene of interest that is complementary to the gRNA, and changing it - either through simple deletion or also insertion via homologous recombination.
• This function could not be realized without encapsulation, because the system would degrade quickly without protection from the environment.
• The motivation to use an SMC instead of engineering the plant directly is that the former would allow for much greater flexibility and responsivity (for example, engineering drought resistance into a crop only if drought is actually imminent). In addition, it is easier to synthesize SMCs than to establish a stable line of genetically engineered seeds.
• The desired outcome is targeted, efficient, and timely genome engineering of plants to introduce traits such as disease-resistance, drought-resistance, flood-resistance, etc.

Components

• Membrane: phospholipids.
• Encapsulated: cell-free tx/tl system, plasmid to express CRISPR-Cas system with gRNA complementary to target plant DNA.
• Tx/Tl system: will need to be bacterial to be complementary with the CRISPR system.
• Communication: the SMC can be taken via vascular transport by the plant through its roots, or, for foliar spray, potentially through the stomata, similarly to the mechanism behind Agrobacterium infiltration. It seems unlikely that a membrane channel would suffice for the transport of an enzyme out of the cell, so we would likely need a way to localize the enzyme to the membrane and induce exocytosis. The exosome could then travel via plasmodesmata to the plant cells.

Experiment Details

• Lipids: I'm really not too sure what lipid would be best; perhaps something resembling the lipids that compose plant exosomes.
• Enzymes: cell-free tx/tl system; vesicle-formation and docking proteins (as I write this I'm getting this feeling that this system is too complicated to be worthwhile, and that I should've pursued my other idea of making a CRISPR-based anti-viral therapeutic SMC instead).
• Genes: Cas enzyme-encoding gene with a membrane-localizing peptide, gRNA
• Host organism: Nicotiana benthamiana, a tobacco plant that easily takes up foreign agents.
• Measurement: Can conduct RNASeq to see if the modified transcripts are being produced by the plant.