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

Abstract

Biocompatibility assessment of nanomaterials has been of great interest due to their potential toxicity. However, conventional biocompatibility tests are 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 for engineering nanomaterials with lower toxicity levels. We have engineered promoters of four heat shock response proteins. As an initial design a reporter coding gene was cloned to downstream of the promoter regions selected. Initial results indicated that native HSP promoter regions were not very promising to generate signals with low background signals. Introducing riboregulators to native promoters eliminated unwanted background signal almost entirely. Unfortunately, this approach also leads a decrease in expected sensor signal. Thus, a repression based genetic circuit, inspired 2 from HSP mechanism of Mycobacterium tuberculosis was constructed. These genetic circuits can report the toxicity of Quantum Dot nanoparticles in one hour with high precision. Our designed nanoparticle toxicity sensors can provide quick reports which can lower the demand for additional experiments with more complex organisms. gene. 10,14 Under steady state conditions, sigma 32 level is maintained at constant levels due to its unstable nature; however, after exposure to any stress, sigma 32 level is dramatically elevated via improved stability as well as increased synthesis. 10,15,16 Sigma 32 is regulated by a negative feedback loop controlled by DnaK-DnaJ-GrpE mechanism. 17 Accumulation of chaperones in this mechanism holds sigma 32 and blocks its activity 18,19 , leading to degradation of sigma 32 by FtsH; a special sigma 32 degrading protease. 20 Therefore, monitoring of HSP levels in cells can be used as a promising stress indicator.Nanomaterials are of great interest for their wide range of applicability across many areas from medicine to optoelectronics. Nanoparticles have size-dependent tunable optical and physical properties; which are not usual for bulk materials 21-23 , nanomaterials are widely used in innovative applications such as in medical diagnostics, drug delivery and targeted photothermal therapy. In all of these approaches patients have to be exposed to nanomaterials. 24 Also, utilization of nanomaterials in consumer goods may contaminate environment, food and textiles. 25 Despite their success in many applications, nanomaterials' high surface-tovolume ratio indicates potential health problems. In addition, due to their small size, nanomaterials are able to penetrate through cellular barriers easily which may cause cellular stress and many adverse effects such as protein unfolding 26 , DNA damage 27,28 , ROS generation 29-31 , and disruption of gene expression 27,28,32 . At the system level, nanomaterials can trigger inflammation and alter immune system response [33][34][35] . Thus, development of a quick and reliable sensor system that reports nanomaterial-triggered toxicity is very critica...

Synthetic biology enables field-deployable biosensors for water contaminants

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Programming living sensors for environment, health and biomanufacturing

“…Synthetic biology offers new toolkits to enhance performance of living sensors for applications in environment, health and biomanufacturing Developments in industrialization have increased dissemination of pollutants and harmful substances which are threatening the environment and human health. Compared to traditional WCBs that use native stress response pathways to report general toxic environment (Kim et al, 2005;Saltepe et al, 2019), synthetic WCBs are able to detect specific pollutants such as heavy metals and metalloids (Wang et al, 2013a;Wan et al, 2019b), organic chemicals and pesticides (Chong and Ching, 2016), waterborne pathogens (Yong and Zhong, 2009) and explosives (Belkin et al, 2017) (Fig. 1).…”

Developing a temperature-inducible transcriptional rheostat inNeurospora crassa

“…The hsp promoters and their expression profiles have been well studied in diverse model organisms, including plants [31,31], mammals [33,34], and fungi [35–38]. In several cases, hsp promoters have been successfully utilized for spatial and temporal control of gene expression [39–41], to promote heat stress tolerance in distinct organisms [42–45], in heterologous protein and chemical production [46,47] or even utilized for synthetic circuits-based biosensors [48].…”