Glycosylation Circuit Enables Improved Catalytic Properties for Recombinant Alkaline Phosphatase.

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins. Here, we engineered a Campylobacter jejuni N-linked glycosylation machinery by overexpressing one of the core glycosylation-related enzymes, PgIB, to increase the glycosylation rate. It has been previously shown that by utilizing N-linked glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. We utilized N-linked glycosylation using an engineered glycosylation pathway to glycosylate a model enzyme, the alkaline phosphatase (ALP) enzyme in Escherichia coli. We have investigated the effects of glycosylation on enzyme properties. Considering the glycosylation mechanism is highly dependent on accessibility of the glycosylation tag, ALP constructs carrying the glycosylation tag at different locations of the gene have been constructed, and glycosylation rates have been calculated. Our results showed that, upon glycosylation, ALP features in terms of thermostability, proteolytic stability, tolerance to suboptimal pH, and denaturing conditions are dramatically improved. The results indicated that the N-linked glycosylation mechanism can be employed for protein manipulation for industrial applications.

A Recombinase-Based Genetic Circuit for Heavy Metal Monitoring.

Rapid progress in the genetic circuit design enabled whole-cell biosensors (WCBs) to become prominent in detecting an extensive range of analytes with promise in many fields, from medical diagnostics to environmental toxicity assessment. However, several drawbacks, such as high background signal or low precision, limit WCBs to transfer from proof-of-concept studies to real-world applications, particularly for heavy metal toxicity monitoring. For an alternative WCB module design, we utilized Bxb1 recombinase that provides tight control as a switch to increase dose-response behavior concerning leakiness. The modularity of Bxb1 recombinase recognition elements allowed us to combine an engineered semi-specific heat shock response (HSR) promoter, sensitive to stress conditions including toxic ions such as cadmium, with cadmium resistance regulatory elements; a cadmium-responsive transcription factor and its cognitive promoter. We optimized the conditions for the recombinase-based cadmium biosensor to obtain increased fold change and shorter response time. This system can be expanded for various heavy metals to make an all-in-one type of WCB, even using semi-specific parts of a sensing system.

Engineering of biofilms with a glycosylation circuit for biomaterial applications.

Glycosylation is a crucial post-translational modification for a wide range of functionalities. Adhesive protein-based biomaterials in nature rely on heavily glycosylated proteins such as spider silk and mussel adhesive proteins. Engineering protein-based biomaterials genetically enables desired functions and characteristics. Additionally, utilization of glycosylation for biomaterial engineering can expand possibilities by including saccharides to the inventory of building blocks. Here, de novo glycosylation of Bacillus subtilis amyloid-like biofilm protein TasA using a Campylobacter jejuni glycosylation circuit is proposed to be a novel biomaterial engineering method for increasing adhesiveness of TasA fibrils. A C. jejuni glycosylation motif is genetically incorporated to tasA gene and expressed in Escherichia coli containing the C. jejuni pgl protein glycosylation pathway. Glycosylated TasA fibrils indicate enhanced adsorption on the gold surface without disruption of fibril formation. Our findings suggest that N-linked glycosylation can be a promising tool for engineering protein-based biomaterials specifically regarding adhesion.

Genetic circuits combined with machine learning provides fast responding living sensors.

Whole cell biosensors (WCBs) have become prominent in many fields from environmental analysis to biomedical diagnostics thanks to advanced genetic circuit design principles. Despite increasing demand on cost effective and easy-to-use assessment methods, a considerable amount of WCBs retains certain drawbacks such as long response time, low precision and accuracy. Here, we utilized a neural network-based architecture to improve the features of WCBs and engineered a gold sensing WCB which has a long response time (18 h). Two Long-Short Term-Memory (LSTM)-based networks were integrated to assess both ON/OFF and concentration dependent states of the sensor output, respectively. We demonstrated that binary (ON/OFF) network was able to distinguish between ON/OFF states as early as 30 min with 78% accuracy and over 98% in 3 h. Furthermore, when analyzed in analog manner, we demonstrated that network can classify the raw fluorescence data into pre-defined analyte concentration groups with high precision (82%) in 3 h. This approach can be applied to a wide range of WCBs and improve rapidness, simplicity and accuracy which are the main challenges in synthetic biology enabled biosensing.

Genetic Circuits To Detect Nanomaterial Triggered Toxicity through Engineered Heat Shock Response Mechanism.

Biocompatibility assessment of nanomaterials has been of great interest due to their potential toxicity. However, conventional biocompatibility tests fall short of providing a fast toxicity report. We developed a whole cell based biosensor to track biocompatibility of nanomaterials with the aim of providing fast feedback to engineer them with lower toxicity levels. We engineered promoters of four heat shock response (HSR) proteins utilizing synthetic biology approaches. As an initial design, a reporter coding gene was cloned downstream of the selected promoter regions. Initial results indicated that native heat shock protein (HSP) promoter regions were not very promising to generate signals with low background signals. Introducing riboregulators to native promoters eliminated unwanted background signals almost entirely. Yet, this approach also led to a decrease in expected sensor signal upon stress treatment. Thus, a repression based genetic circuit, inspired by the HSR mechanism of Mycobacterium tuberculosis, was constructed. These genetic circuits could report the toxicity of quantum dot nanoparticles in 1 h. Our designed nanoparticle toxicity sensors can provide quick reports, which can lower the demand for additional experiments with more complex organisms.