Design and Construction of a Whole Cell Bacterial 4-Hydroxyphenylacetic Acid and 2-Phenylacetic Acid Bioassay

Abstract

IntroductionAuxins are hormones that regulate plant growth and development. To accurately quantify the low levels of auxins present in plant and soil samples, sensitive detection methods are needed. In this study, the design and construction of two different whole cell auxin bioassays is illustrated. Both use the auxin responsive element HpaA as an input module but differ in output module. The first bioassay incorporates the gfp gene to produce a fluorescent bioassay. Whereas the second one utilizes the genes phzM and phzS to produce a pyocyanin producing bioassay whose product can be measured electrochemically.ResultsThe fluorescent bioassay is able to detect 4-hydroxyphenylacetic acid (4-HPA) and 2-phenylacetic acid (PAA) concentrations from 60 μM to 3 mM in a dose-responsive manner. The pyocyanin producing bioassay can detect 4-HPA concentrations from 1.9 to 15.625 μM and PAA concentrations from 15.625 to 125 μM, both in a dose-responsive manner.ConclusionA fluorescent whole cell auxin bioassay and an electrochemical whole cell auxin bioassay were constructed and tested. Both are able to detect 4-HPA and PAA at concentrations that are environmentally relevant to plant growth.

Regulation of indole‐3‐acetic acid biosynthesis and consequences of auxin production deficiency in Serratia plymuthica

“…Under the conditions tested, we were unable to observe IAA binding by HpaA A153 or HpaR A153 (Table S4). Furthermore, HpaA A153 or HpaR A153 did not recognize PAA (Table S4), an auxin that induces the expression of the hpa cluster in E. coli and that was suggested to be a HpaA ligand (Dierckx et al, 2015;Prieto & García, 1997). b Genes that were included due to the general down-regulation of the paa phenylacetic acid catabolic operon at least 1.5-fold.…”

Cell-Based Biosensor with Dual Signal Outputs for Simultaneous Quantification of Phenylacetic Acid and Phenylethylamine

“…13 The quantification of auxin for horticulture purposes requires rapid and sensitive analysis of plant tissues, soil, and environmental aqueous samples. The analytical methods, such as liquid chromatography in combination with ESI-mass spectrometry (LC−ESI-MS), 14 GC−MS, 15 and isocratic reversed-phase HPLC with precolumn derivatization were developed for the quantification of auxin; 16 24 however, it responded to both PA and its hydroxylated derivatives. Consequently, our primary objective in this study was to develop a method for the simple, rapid, and sensitive detection and differentiation of PA and PEA via bacterial catabolic regulons.…”

“…The analytical methods, such as liquid chromatography in combination with ESI-mass spectrometry (LC–ESI-MS), GC–MS, and isocratic reversed-phase HPLC with precolumn derivatization were developed for the quantification of auxin; however, these methods require expensive instruments and highly specialized technicians. The molecule-based or cell-based biosensors have attracted a lot of research effort and have been used in environmental monitoring and clinical diagnostics. − Dierckx et al recently developed a whole-cell biosensor for the detection of environmental auxin based on bacterial hpa regulon; however, it responded to both PA and its hydroxylated derivatives. Consequently, our primary objective in this study was to develop a method for the simple, rapid, and sensitive detection and differentiation of PA and PEA via bacterial catabolic regulons.…”

Amperometric Detection of the Urinary Disease Biomarker p-HPA by Allosteric Modulation of a Redox Polymer-Embedded Bacterial Reductase

“…Moreover, fast-scan cyclic voltammetry requires special potentiostats, low-noise electrochemical setups and microelectrodes and a high level of operator expertise, rendering such applications unpractical for clinical diagnostics. A recent whole-cell biochemical assay with electrochemical readout involved the voltammetric detection of redox-active pyocyanine, the cellular production rate of which is controlled by p -HPA, enabling indirect determination of the target biomarker but with rather low sensitivity and selectivity …”

“…A recent whole-cell biochemical assay with electrochemical readout involved the voltammetric detection of redox-active pyocyanine, the cellular production rate of which is controlled by p-HPA, enabling indirect determination of the target biomarker but with rather low sensitivity and selectivity. 17 Existing literature examples and successful commercial applications are good evidence that amperometric biosensors with enzymes embedded in polymer layers offer promising selectivity and sensitivity. 18 In the case of biocompatible protein immobilization in adapted hydrophilic redox polymers, biosensors are known to be stable during storage and they can be operated entirely without reagents, which are important characteristics for the construction of a miniaturized device for easy point-of-care biosensing.…”