**4.** *E. coli***-based biosensors: tools for many applications**

In biotechnology, biosensors are broadly defined as any device based on biological part, cell, tissue, or protein complex that are linked to a mechanical sensor or analytical receptor that provides a measurable signal proportional to the analyte in the reaction [66, 67]. *E. coli*-based biosensors using plasmid or chromosomal constructs are useful for the detection of environmental traits or hazards or measuring cellular processes as any standard reporter system [68–70].

In **Figure 1**, we depict the basic design for whole-cell biosensors and some applications. Plasmid vectors with all the possible modifications can lead to almost endless combinations. For practical applications, there are commercial vectors that can be used for such purposes or as mentioned in the previous sections, plasmid methods are powerful enough for fast and robust biosensor design.

**Figure 1.** Basic principles for biosensor design. First, the proper design and the experimental creation of reporter strains. With current knowledge, either plasmid creation or whole genome engineering can lead to the creation of a reporter strain. Second, incorporation of such elements into the host cell. Third, sensing module and response measurement. With current reporter proteins and detection technology, it is relatively easy to generate biosensors that can be used in different applications with high sensitivity and selectivity.

The basic design considers the following: copy number, reporter proteins, detection methods, and control elements. The latter is basically the most important feature. As shown in **Table 1**, the available databases provide enough information for promoter selection and design. Bioinformatic tools can make this process easier [71]. Also, generation and detection of this kind of biosensors are cost-effective and easy to generate and reasonably sensitive [71]. In terms of speed, sample analysis with whole-cell biosensors is fast and cheap in comparison with analytical methods. The sensitivity of analytical methods is higher and more accurate, but biosensors are a good alternative for fast detection of hazards. Also, they can be coupled with the controlled production of metabolites of commercial importance.

observations from the synthetic genomes, we envision a bright future for bacterial molecular genetics in many fields of biotechnology, such as the production of molecules for human

*E. coli* is an extensively studied organism, with all the cumulative data we can ensure that with all this knowledge, we can design tools. In the following section, we comment on the

In biotechnology, biosensors are broadly defined as any device based on biological part, cell, tissue, or protein complex that are linked to a mechanical sensor or analytical receptor that provides a measurable signal proportional to the analyte in the reaction [66, 67]. *E. coli*-based biosensors using plasmid or chromosomal constructs are useful for the detection of environmental traits or hazards or measuring cellular processes as any standard reporter system [68–70].

In **Figure 1**, we depict the basic design for whole-cell biosensors and some applications. Plasmid vectors with all the possible modifications can lead to almost endless combinations. For practical applications, there are commercial vectors that can be used for such purposes or as mentioned in the previous sections, plasmid methods are powerful enough for fast and

**Figure 1.** Basic principles for biosensor design. First, the proper design and the experimental creation of reporter strains. With current knowledge, either plasmid creation or whole genome engineering can lead to the creation of a reporter strain. Second, incorporation of such elements into the host cell. Third, sensing module and response measurement. With current reporter proteins and detection technology, it is relatively easy to generate biosensors that can be used in

**4.** *E. coli***-based biosensors: tools for many applications**

262 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

wellbeing.

biosensors that are *E. coli*-based.

robust biosensor design.

different applications with high sensitivity and selectivity.

In the literature, there are several reports where *E. coli*-based biosensors have been successful for detecting different traits: oxidants [72, 73], DNA damaging compounds [74], membrane-damaging compounds [75], protein-damaging compounds [76], aromatic compounds [77–79], xenobiotics [80], antibiotic panels using reporter strains without antibiotic selection [81], etc. The only limitation is the available sensor module and the design. The reporter protein is also important. Stability and reproducibility are two important aspects of biosensor design. In our experience, Green Fluorescent protein (GFP) protein is superior to luciferase, especially that we can detect GFP by various methods (we find flow cytometry, fluorometry, and confocal microscopy our top preferences) without cell lysis or substrate mixtures that are time-consuming [82].

With the improvement of DNA synthesis, recoding protein-coding genes for the desired function is expanding the capabilities of transcription factors, and reporter proteins have created novel sensor modules. For example, XilR recoding has led to a sensor that can detect millimolar concentrations of trinitrotoluene and its derivative compounds [83]. By using shuttle vectors, we can generate biosensors that we can transfer from one host to another, which can provide information about differences in physiological responses during the exposure to a given environmental trait.

*E. coli* plasticity and tools such as BioBrick building (using standardized DNA fragments with compatible ends for fast assembly) can facilitate plasmid and reporter constructs [84, 85]. Correlations of cell growth and physiology with expression patterns from reporter constructs can expand our knowledge of the impact of exposure to the external stimulus on cell physiology. Biosensors based on whole cells are a cheap alternative and can be coupled to portable devices. Using qualitative reporters can be applied in field research [70]. One good example is the detection of parasites without using cold-protected samples or complicated equipment for the detection process [86].

In the following section, we provide our final overview of the impact of *E. coli* in the synthetic biology future.
