Results

Introduction

Our team focused on engineering Acinetobacter baylyi (ADP1) to function as a reliable detector of environmental DNA (eDNA) by exploiting its natural competency. We focused our engineering efforts on ADP1-ISx, a transposon-free, naturally competent derivative of ADP1 that was previously characterized by the 2022 UT Austin iGEM team.¹ We designed a sensor to integrate into ADP1's genome to recognize eDNA released by microcystin-producing cyanobacteria. Specifically, our sensor targets segments of the mcy gene cluster, the group of genes responsible for microcystin synthesis in Microcystis aeruginosa.² In addition to sensing eDNA, we aimed to increase the recombination frequency by introducing a plasmid encoding a recombinase gene. This would increase transformation frequency at shorter homology flanks, which will be useful when detecting partially degraded eDNA. Finally, we worked to incorporate the mlrA and mlrD genes into ADP1's genome with inducible promoters characterized by our microcystin production team, enabling potential degradation of the microcystin toxin.³

To accomplish these aims, our team divided into four main subgroups:

• 1. Sensor integration and validation in ADP1.
  1. Inserted a DNA-based sensor into ADP1's genome.
  2. Verified genomic integration via colony PCR and sequencing.
  3. Demonstrated that the sensor remains inactive until recombination with the target sequence occurs.
  4. Confirmed functionality at low DNA concentrations, plasmid DNA, genomic DNA, and within mixed media.
• 2. Enhancing recombination efficiency using recombinase.
  1. Introduced a recombinase-encoding plasmid to ADP1.
  2. Quantified the resulting increase in recombination frequency, particularly at shorter homology lengths.
• 3. Incorporation of microcystin-degrading genes into sensor construct.
  1. Constructed part plasmids of mlrA and mlrA+mlrD for potential inclusion in future sensor designs.
• 4. Demonstrating large-scale genome engineering in ADP1.
  1. Designed part plasmids containing inducible promoters and GFP as proof-of-concept modules.
  2. Constructed of a custom backbone containing ADP1 homology flanks and type IIS restriction sites (BsaI and BsmBI) to expedite future sensor assembly.

Figure 1. How each objective relates to the engineering of ADP1. Created with Biorender.com