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Since the PureExpress Cell Free mix is so expensive, I was wondering if it might be possible to just dilute the mix to increase the number of reactions we need to use.

From this image I found:

DIY Cell Free Reactions

It seems like by just adding more 3PGA, amino acids we should be able to get more reactions out of a diluted solution. From my understanding, this would mean that 3PGA and the amino acids are the only consumables here. Would diluting just change the amount time it takes for the reaction to occur or is there something else that I'm missing here.

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  • $\begingroup$ Really interesting question, but your image seems to show a crude cell free system and not a pure cell free system, unless I’m misunderstanding something! $\endgroup$ – Brad0440 Mar 26 at 22:51
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    $\begingroup$ Yeah, this was the best picture I could find to represent my understanding. $\endgroup$ – rkrishnasanka Mar 27 at 15:28
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Try using this equivalent thing you can make in lab yourself for way cheaper. The protocol is a pain in the a** -- I've done it myself several times and it takes 2 people working in tandem for a long week -- but it does work. (The first author of this paper has gone on to create a successful company based on this exact cell-free system, Sutro BioPharma) https://www.jove.com/t/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free

If you're at an academic institution and interested in trying this, I can walk you through it, some modifications that make it more efficient, and which parts have some "wiggle" room.

I would not recommend diluting PURExpress... Every component in cell-free reactions is essential and carefully calibrated. Modifying any of them can result in dramatically decreased efficiency. For example, would you be able to adjust all the ion concentrations as you dilute the mixture?

I recommend using minimum 5 µL volumes in a 384-well plate.

also good luck buying 3pga... there was a massive shortage for at least a year when i was doing this stuff... maybe it's better now.

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  • $\begingroup$ Nice answer! It sounds like you have some good synbio experience. Would you like to join the SynBio SE site proposal? $\endgroup$ – jakebeal Mar 27 at 8:28
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The PURExpress system from NEB is incredibly expensive, for this reason I have avoided it all costs during my experiments with cell-free systems.

This is the paper I've used to create my own cheaper cell-free systems while also discussing reasons why using the NEB PURExpress system is not recommended: https://pubs.acs.org/doi/10.1021/acssynbio.8b00427

  • "Although NEB provides a few different formulations of the PURE system, the commercial system can’t be customized or optimized by the user, and the precise formulation of the commercial PURE system is not publicly available."

This might answer your question as to why I would not dilute the PURExpress system since the precise formulation is not publicly known. Thus, we can hypothesize what would work at various concentrations, but at the end of the day it is unknown what they specifically use in this reaction and how one could optimize it for their own purposes. It would take some trial and error if you really wanted to dilute/change the formulation.

  • "Our method co-cultures and induces all 36 protein producing E. coli clones in a single flask followed by a single Ni-NTA purification."

This is the method I personally use and it can be time intensive, however the payoff is the cost and ability to optimize your system for your specific experiment.

  • "The PURE system consists of several different components [7], that can be separated into three main categories: proteins (transcription, translation, and energy regeneration),ribosomes, and small molecule components (salts, buffers, NTPs, creatine phospate, and folinic acid)

If you were to go down the dilution route, I would suggest reaching out to NEB before hand to receive consultation for the purposes of your experiment and any consumables you may need to also purchase. This quote from the paper shows how your system has many considerations for consumables you may need to further research to maximize the expression of certain proteins. As for how this affects the reaction time, dilution shouldn't affect time, it will however affect the amount of product you get out of your reaction.

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  • $\begingroup$ I guess in a macroscale, it wouldn't really affect the reaction time but in the scenario that there are reusable components then shouldn't it affect the reaction rate? (Thinking back to high-school physical chemistry) $\endgroup$ – rkrishnasanka Mar 27 at 15:31

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