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EngineeringSuccess

Introduction

At its core, the engineering success of CyanoSense was centered on the manipulation of Acinetobacter baylyi (ADP1), a bacterial chassis known for its high natural competency to recombine with homologous DNA.1 We leveraged this property to engineer a DNA-based biosensor2 in ADP1 capable of detecting environmental DNA (eDNA) from microcystin-producing cyanobacteria. Upon recognizing a target DNA sequence, the sensor will undergo homologous recombination, restoring a disrupted reading frame and activating a downstream chloramphenicol resistance gene.

To target microcystins, hepatotoxins produced by Microcystis aeruginosa,3 our sensor was designed to recognize the genes that produce microcystins. To improve recombination efficiency, we also introduced a recombinase enzyme system,4 hypothesizing that it could enhance transformation frequency and sensor responsiveness.

Over the course of this project, our team achieved four key engineering milestones:

  1. Correct cloning and assembly of the linear DNA biosensor
  2. Accurate insertion of the DNA sensor into the ADP1 chromosome
  3. Successful recognition, recombination, and activation of chloramphenicol resistance upon sensing the target DNA
  4. Demonstration of improved recombination efficiency through the recombinase system

Each success reflects a full iteration of the Design → Build → Test → Learn engineering cycle, detailed below.

Image

Figure 1. Design-Build-Test-Learn cycle for ADP1 sensor and recombinase engineering.

References

  1. Santala, Suvi, and Ville Santala. “Acinetobacter Baylyi ADP1-Naturally Competent for Synthetic Biology.” Essays in Biochemistry, vol. 65, no. 2, Jul. 2021, pp. 309-18. PubMed, https://doi.org/10.1042/EBC20200136.
  2. Hua, Yu, et al. “DNA-Based Biosensors for the Biochemical Analysis: A Review.” Biosensors, vol. 12, no. 3, Mar. 2022, p. 183. PubMed, https://doi.org/10.3390/bios12030183.
  3. Melaram, Rajesh, et al. “Microcystin Contamination and Toxicity: Implications for Agriculture and Public Health.” Toxins, vol. 14, no. 5, May 2022, p. 350. PubMed, https://doi.org/10.3390/toxins14050350.
  4. Wang, Yueju, et al. “Recombinase Technology: Applications and Possibilities.” Plant Cell Reports, vol. 30, no. 3, Mar. 2011, pp. 267-85. PubMed, https://doi.org/10.1007/s00299-010-0938-1.
  5. Zheng, Yanli, et al. “Reconstitution and Expression of Mcy Gene Cluster in the Model Cyanobacterium Synechococcus 7942 Reveals a Role of MC-LR in Cell Division.” New Phytologist, vol. 238, no. 3, May 2023, pp. 1101-14. DOI.org (Crossref), https://doi.org/10.1111/nph.18766.
  6. Chuong, Jeffrey, et al. “Engineered Acinetobacter Baylyi ADP1-ISx Cells Are Sensitive DNA Biosensors for Antibiotic Resistance Genes and a Fungal Pathogen of Bats.” ACS Synthetic Biology, vol. 14, no. 7, Jul. 2025, pp. 2488-93. DOI.org (Crossref), https://doi.org/10.1021/acssynbio.5c00360.
  7. Tucker, Ashley T., et al. “Defining Gene-Phenotype Relationships in Acinetobacter Baumannii through One-Step Chromosomal Gene Inactivation.” mBio, edited by Louis M. Weiss, vol. 5, no. 4, Aug. 2014, pp. e01313-14. DOI.org (Crossref), https://doi.org/10.1128/mBio.01313-14.