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I'm looking to phase-separate the expression of two enzymes and hence am looking for a multi-component genetic oscillator. However, repressilators and metabolators have a large period of around 45 mins. Smolen oscillators as described in Stricker et al 2008 A fast, robust and tunable synthetic gene oscillator seem like something I could use, however, I'm not sure if I should, I am new to oscillators.

Please let me know what kind of oscillator you would recommend for a 2-20 min period time, preferably robust. Any other relevant information or recommendations are welcome. The intended system is Synechocystsis 6803 but initial work may be done in E coli.

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I believe that what you are aiming to do is not possible with the mechanisms that you propose.

In most transcriptional and translational genetic circuits, the limiting factor for switching state is dilution or decay rate.

This is because genetic regulatory networks directly control rate of production, rather than concentration. Thus, even if your oscillator instantaneously switches on and off (which it won't), your ability to turn off an enzyme depends on the rate at which its concentration decreases.

Degradation mechanisms in the cell do not operate at the time scales that you're looking for (see, for example [1],[2]), so you need to depend on dilution. E. coli at full stretch goes at about at 20 minute division time, and even the fastest doubling organisms (e.g., V. naturiegens) max out at around 7-10 minute doubling time. That gives you a half-life, not complete removal, so if you want an enzyme to be mostly off (not just decreased some), then your target times are simply not achievable with genetic regulation.

When cells need to regulate protein activity on these tighter time scales, they generally regulate protein state rather than protein concentration. This is more difficult, but if you really need to hit such tight times, I recommend that you look into such mechanisms. Alternately, do you really need to modulate this quickly?

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  • $\begingroup$ I see. I understand the limitation my approach has. Thank you for your detailed answer. I'm actually not sure what period we're looking for, will have to check that out through mathematical modelling. I'll look into what you mentioned. Enzyme 1's product (P) has a feedback inhibition effect on Enzyme 2, and we want to separate their concentrations in time. Are oscillators a good approach or would placing Enzyme 2 under transcriptional control of a P sensitive transcription factor? Any other approaches you can think of? $\endgroup$
    – Siddharth Fitwe
    Jul 19 '21 at 12:23
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    $\begingroup$ Oscillators is a very interesting idea, if you can afford a longer time-scale. Available oscillators are fragile, though, so doing that would likely entail significant focus on the oscillator. You could also use transcriptional control or try to do spatial separation (e.g., two separate strains) if the product can be exported. $\endgroup$
    – jakebeal
    Jul 19 '21 at 13:55

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