Rational design and characterization of nitric oxide biosensors in E. coli Nissle 1917 and Mini SimCells

Xiaoyu J. Chen, Baojun Wang, Ian P. Thompson, Wei E. Huang

Research output: Contribution to journalArticlepeer-review

Abstract

Nitric oxide (NO) is an important disease biomarker found in many chronic inflammatory diseases and cancers. A well-characterized nitric sensing system is useful to aid the rapid development of bacteria therapy and synthetic biology. In this work, we engineered a set of NO-responsive biosensors based on the PnorV promoter and its NorR regulator in the norRVW operon; the circuits were characterized and optimized in probiotic Escherichia coli Nissle 1917 and mini SimCells (minicells containing designed gene circuits for specific tasks). Interestingly, the expression level of NorR displayed an inverse correlation to the PnorV promoter activation, as a strong expression of the NorR regulator resulted in a low amplitude of NO-inducible gene expression. This could be explained by a competitive binding mechanism where the activated and inactivated NorR competitively bind to the same site on the PnorV promoter. To overcome such issues, the NO induction performance was further improved by making a positive feedback loop that fine-tuned the level of NorR. In addition, by examining two integration host factor (IHF) binding sites of the PnorV promoter, we demonstrated that the deletion of the second IHF site increased the maximum signal output by 25% (500 μM DETA/NO) with no notable increase in the basal expression level. The optimized NO-sensing gene circuit in anucleate mini SimCells exhibited increased robustness against external fluctuation in medium composition. The NO detection limit of the optimized gene circuit pPnorVβ was also improved from 25.6 to 1.3 nM in mini SimCells. Moreover, lyophilized mini SimCells can maintain function for over 2 months. Hence, SimCell-based NO biosensors could be used as safe sensor chassis for synthetic biology.
Original languageEnglish
Pages (from-to)2566–2578
Number of pages13
JournalACS Synthetic Biology
Volume10
Issue number10
Early online date22 Sep 2021
DOIs
Publication statusPublished - 15 Oct 2021

Keywords

  • synthetic biology
  • mini SimCell
  • minicells
  • nitric oxide biosensors
  • NorR regulatory circuits

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