Digitalizing heterologous gene expression in Gram‐negative bacteria with a portable ON/OFF module

Abstract

While prokaryotic promoters controlled by signal‐responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription—as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation‐inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.

Standardization of inducer-activated broad host range expression modules: Debugging and refactoring an alkane-responsive AlkS/PalkBdevice

“…3A). Although this level is not too high (it remains within the same order of magnitude than the baseline expression), it has to be taken into account when expression of toxic proteins or coupling with other devices are pursued (27). A second aspect of the results shown in Fig.…”

Industrial biotechnology of Pseudomonas putida: advances and prospects

“…Furthermore, rrn operons have demonstrated a strong preference for biosynthetic gene cluster integration in P. putida , due to stable integration and strong expression by the native promoters (Domröse et al 2019 ). Finally, it was recently demonstrated that fine-tuning of metabolic pathways via targeted proteolysis enables a new control layer of engineered pathways (Calles et al 2019 ; Volke et al 2020b ) .…”

Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology

“…The radius described by the partially formed monomers is r 1 and r 2 respectively. In this scenario, the spheres are then described by: (9) and (x − ∆x) 2 + y 2 + z 2 ≤ r 2 2…”

“…The development of complex mathematical modelling techniques [4,5], along with the advances in DNA synthesis and assembly [6], allows for the making of new-to-Nature networks of regulatory proteins-the so-called genetic circuits-with increasing information processing abilities. Relatively simple models of computation, like combinatorial [7] and sequential logic [8], have been successfully implemented in organisms such as bacteria [9], yeasts [10] or even mammalian cells [11]. Examples of these genetic circuits include logic gates [12,13], multiplexers [14], half-adders [15], counters [16] and memories [17]-even more complex processes such as analogue [18,19] and distributed computations [20,21,22].…”