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 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.

Genetic Circuits Combined with Machine Learning Provides Fast Responding Living Sensors

“…In our engineered bacterial WCB design, we constructed a complex and tightly controlled gold detecting circuit combining a semi-specific stress biosensor based on heat shock response (HSR) 60 and a specific biosensor for gold sensing 61 . In the circuit, a constitutively expressed HSR repressor, HspR, blocks gene expression from its cognitive promoter, P dnaK-IR3-IR3 , controlling the expression of gold specific transcription factor, GolS, and a site-specific recombinase, Bxb1.…”

“…To construct sensing and output modules, inverted PgolB promoter with Bxb1 recognition sites and reporter-terminator (GFP-rrnBT1) pair were amplified by polymerase chain reaction (PCR) using Q5 High-Fidelity DNA Polymerase (M0491, NEB) in thermal cycler (C1000 Touch, Bio-Rad). For backbone, formerly constructed mProD HspR pET22b vector 60 was linearized with SpeI restriction endonuclease enzyme (R3133, NEB). To construct actuating module, Bxb1 recombinase and GolS were amplified by PCR.…”

“…To construct actuating module, Bxb1 recombinase and GolS were amplified by PCR. For backbone, formerly constructed PdnaK-IR3-IR3 GFP pZa vector 60 was digested with MluI restriction endonuclease enzyme (R3198, NEB) to exclude gfp from the vector. All pieces were run on 1 or 2% (w/v) Agarose gel stained with SYBR Safe DNA gel stain (S33102, Invitrogen).…”

The Emerging Trend of Bio-Engineering Approaches for Microbial Nanomaterial Synthesis and Its Applications

“…The synthetic gene circuit has also been used to detect the cellular toxicity of nanomaterials. A synthetic gene circuit consisting of four heat shock promoter regions along with a gene sequence coding for reporter protein acts as the biosensor for the nanoparticle induce toxicity (Saltepe et al, 2019). Such synthetic biosensor circuit based on use of HSP system can be applied to assess the toxicity of other types of nanomaterials as well.…”

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).…”