Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli

Abstract

Genetically programmed circuits allowing bifunctional dynamic regulation of enzyme expression have far-reaching significances for various bio-manufactural purposes. However, building a bio-switch with a post log-phase response and reversibility during scale-up bioprocesses is still a challenge in metabolic engineering due to the lack of robustness. Here, we report a robust thermosensitive bio-switch that enables stringent bidirectional control of gene expression over time and levels in living cells. Based on the bio-switch, we obtain tree ring-like colonies with spatially distributed patterns and transformer cells shifting among spherical-, rod- and fiber-shapes of the engineered Escherichia coli. Moreover, fed-batch fermentations of recombinant E. coli are conducted to obtain ordered assembly of tailor-made biopolymers polyhydroxyalkanoates including diblock- and random-copolymer, composed of 3-hydroxybutyrate and 4-hydroxybutyrate with controllable monomer molar fraction. This study demonstrates the possibility of well-organized, chemosynthesis-like block polymerization on a molecular scale by reprogrammed microbes, exemplifying the versatility of thermo-response control for various practical uses.

Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation

“…This implied a larger pool of unbounded NT7 and CT7 protein fragments would be readily available for reconstitution in the upcoming activation phase and led to the estimated half-activation response within tens of minutes. This suggests that the thermally responsive characteristic of Thermal-T7RNAP system is potentially useful in temperature-based dynamic regulation of bio-processes for example in bioproduction relevant range (30 -37 °C) such as in the control of metabolic fluxes (Harder et al, 2018;Wang et al, 2021), that can complement with existing light-based systems (Zhao et al, 2019;Zhao et al, 2018). During the activation phase, when the temperature alternated between 30 °C and 40 °C (Supplementary Figure S6c,d), the initial spike in GFP synthesis rate could be attributed to the faster initial increase in cell growth before growth stabilisation (data not shown), although the artefact was not as apparent when alternating between 30 °C and 37 °C.…”

“…Taken together, the Thermal-T7RNAPs has expanded the synthetic biology thermal control toolkit for biomedical (Abedi et al, 2020;Gamboa et al, 2020;Miller et al, 2018;Piraner et al, 2017) and industrial applications (Harder et al, 2018;Rodrigues et al, 2017;Salila Vijayalal Mohan et al, 2020;Wang et al, 2021;Xu et al, 2020) by providing an alternative mode of thermal regulation. The Thermal-T7RNAPs can be an useful addition for combinatorial multi-level temperature-based gene regulations (e.g.…”

Optimization of Nanoparticles for Smart Drug Delivery: A Review

“…For example, precursors are now common in nervous system drugs, antitumor system drugs, and antiviral drugs [98]. These precursor drugs, which are not effective in vitro, can be activated by the target micro-environment after entering the human body to play a therapeutic role [99]. An exogenous stimulus condition refers to the type of robot that is deformed after receiving external stimuli [95].…”

Toward Multiplexed Optogenetic Circuits

“…In particular, a number of intra- and extracellular stimuli are perceived by a set of cellular sensor arrays. The internal stimuli rely on the change in the internal cellular state such as cellular burden and intracellular metabolites ( Dahl et al, 2013 ; Ceroni et al, 2018 ), whereas the external stimuli sense environment signals including pH, oxygen, temperature, light, and small chemical inducer ( Fernandez-Rodriguez et al, 2017 ; Moser et al, 2018 ; Meyer et al, 2019 ; Wang et al, 2021 ). Subsequently, these signals are further transmitted and potentially integrated to activate or repress complex cellular signaling cascades or genetic regulatory circuits.…”