A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts.

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.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

BACKGROUND: Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. RESULTS: For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. CONCLUSIONS: The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

Experimental Study on the Mechanical Properties of Disposable Mask Waste-Reinforced Gangue Concrete.

This paper is grounded on the following information: (1) Disposable masks primarily consist of polypropylene fiber, which exhibits excellent flexibility. (2) China has extensive coal gangue deposits that pose a significant environmental hazard. (3) Coal gangue concrete exhibits greater fragility compared to regular concrete and demonstrates reduced resistance to deformation. With the consideration of environmental conservation and resource reutilization, a preliminary concept suggests the conversion of discarded masks into fibers, which can be blended with coal gangue concrete to enhance its mechanical characteristics. In this paper, the stress-strain law of different mask fiber-doped coal gangue concrete (DMGC) under uniaxial compression is studied when the matrix strength is C20 and C30, and the effect of mask fiber content on the mechanical behavior and energy conversion relationship of coal gangue concrete is analyzed. The experimental results show that when the content of mask fiber is less than 1.5%, the strength, elastic modulus, deformation resistance, and energy dissipation of the concrete increase with mask fiber content. When the amount of mask fiber is more than 1.5%, because the tensile capacity and energy dissipation level of concrete produced by the mask fiber cannot compensate for the compression and deformation resistance of concrete of the same quantity and because excess fiber is difficult to evenly mix in the concrete, there are pore defects in concrete, which decreases the concrete strength due to the increase in mask fiber. Therefore, adding less than 1.5% mask fiber helps to improve the ductility, toughness, impermeability, and oxidation and control the cracking of coal gangue concrete. Based on Weibull theory, a constitutive model of DMGC is established, which fits well with the results of a uniaxial test, providing support for understanding the mechanical law of mask fiber-doped concrete.