Functional Characterisation of Engineered Bacterial Biosensors for Kynurenine Detection
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The kynurenine (KYN) pathway is the major catabolic pathway for tryptophan in humans, producing several metabolites that influence health. In clinical settings, KYN levels serve as a valuable biomarker for diagnosis and prognosis of inflammatory and neurological diseases. Nevertheless, KYN detection relies on mass spectrometry analysis, which requires specialised knowledge and expertise with high operational costs. The bacterial biosensor presents as a promising tool for rapid and cost-effective targeted substance detection due to its ease of genetic modifications. Therefore, this study aimed to develop an engineered bacterial biosensor by integrating a genetic module in a plasmid designed for KYN detection harboured in an Escherichia coli chassis. The KYN biosensing component in the genetic module encodes a KYN pathway regulator (KynR) from Pseudomonas aeruginosa, driven by the PBAD arabinose-inducible promoter. Upon expression, KynR would bind to the exogenous KYN and the bacterial responding kyn promoter to express the downstream green fluorescent protein gene to emit a fluorescence signal. However, despite successful induction by arabinose and the presence of KYN, biosensors with different gene orientation and genetic components failed to produce a significant fluorescence signal. These findings suggest that the sensitivity of P. aeruginosa KynR is insufficient to detect physiological level of KYN. Further exploration of alternative biological sensing components is warranted.
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Thank you very much for submitting your manuscript to Access Microbiology. I have had a preliminary editorial review through it before sending it to peer review. I would like to request some clarifications and revisions before sending it our to review. Could you please provide a revised manuscript addressing the below? - Writing style: currently the manuscript is a bit hard to follow as the style is very direct and the may experience difficulties for understanding the rationale behind the experimental design and the purpose of the experiments. Please revise this accordingly. - Knoten et al (2011), which is cited in the manuscript, described a promoter region for kynA and kynB than is directly bound by KynR. Please describe how these promoter regions compare to those used in the design of the proposed biosensors. - GFP fluorescence …
Thank you very much for submitting your manuscript to Access Microbiology. I have had a preliminary editorial review through it before sending it to peer review. I would like to request some clarifications and revisions before sending it our to review. Could you please provide a revised manuscript addressing the below? - Writing style: currently the manuscript is a bit hard to follow as the style is very direct and the may experience difficulties for understanding the rationale behind the experimental design and the purpose of the experiments. Please revise this accordingly. - Knoten et al (2011), which is cited in the manuscript, described a promoter region for kynA and kynB than is directly bound by KynR. Please describe how these promoter regions compare to those used in the design of the proposed biosensors. - GFP fluorescence measurement are not usually done directly in LB broth, as this medium has autofluorescence at a similar wavelength. Instead, samples are normally grown in 10% LB broth or cells are harvested and resuspended in PBS. Do the authors think this may be the reason why the LB control produces the same fluorescence as the biosensor samples (Figure 4)? Please provide a revised version of the manuscript addressing these issues before progressing to peer review.
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