Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.
Summary Hazardous materials, such as heavy metals, are the major sources of health risk. Using genetically modified organisms (GMOs) to dispose heavy metals has the advantages of strong environmental compatibility and high efficiency. However, the biosecurity of GMOs used in the environment is a major concern. In this study, a self‐controlled genetic circuit was designed and carefully fine‐tuned for programmable expression in Pseudomonas putida KT2440, which is a widely used strain for environmental bioremediation. The cell behaviours were controlled by automatically sensing the variation of Hg2+ concentration without any inducer requirement or manual interventions. More than 98% Hg2+ was adsorbed by the engineered strain with a high cell recovery rate of 96% from waterbody. The remaining cells were killed by the suicide module after the mission was accomplished. The escape frequency of the engineered P. putida strain was lower than 10−9, which meets the recommendation of US NIH guideline for GMOs release (<10−8). The same performance was achieved in a model experiment by using natural lake water with addition of Hg2+. The microbial diversity analysis further confirmed that the remediation process made little impact on the indigenous ecosystem. Thus, this study provides a practical method for environmental remediation by using GMOs.
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