Terephthalate biosensors and uses thereof

Inventors

Jha, Ramesh K. • Dale, Taraka T. • Bermel, Ryan E. • NEIDLE, Ellen Lee

Assignees

University of Georgia Research Foundation Inc • Triad National Security LLC

Abstract

Biosensors for detection terephthalic acid (TPA) and methods of their use are provided. The biosensors include a nucleic acid encoding a TphR protein, a promoter regulated by TPA, and a reporter operably linked to the promoter. Vectors and host cells including the biosensors are also provided.

Core Innovation

The invention provides a terephthalic acid (TPA) biosensor comprising a nucleic acid encoding a TPA responsive transcription factor from Comamonas testosteroni (TphR) together with a TphR regulated promoter. The TphR regulated promoter comprises the nucleic acid sequence of nucleotides 4-114 of SEQ ID NO: 12, and a nucleic acid encoding a reporter protein is operably linked to the promoter to generate a readout associated with TPA responsiveness.

In the disclosed embodiments, the reporter protein includes a fluorescent protein, and the sensor-reporter system is configured so that TPA responsiveness is detected through the reporter operably linked to the TphR regulated promoter. Additional embodiments constrain nucleic acid sequence content using defined sequence identity relationships, including at least 95% sequence identity to nucleotides 2862-4457 of SEQ ID NO: 1 or its reverse complement.

The document further describes implementation contexts for the biosensor, including vectors and host cells. In particular, the host cell is specified as a bacterial cell that is an Acinetobacter baylyi cell, and further as an Acinetobacter baylyi ADP1 cell. The disclosed system is characterized through promoter diversification into multiple generations (pTPA1–pTPA3), and it includes improved detection under acidic pH and ratiometric/background control using sfCherry copy number with FACS-based sorting.

Claims Coverage

The document contains two independent claims that cover a TPA biosensor built from a Comamonas testosteroni TphR transcription factor, a specifically defined TphR regulated promoter subsequence, and an operably linked reporter protein nucleic acid, and a nucleic acid defined by the same promoter subsequence. Overall, the claim set is structured around three core inventive elements: the TphR-based TPA responsive regulatory system, the promoter defined by nucleotides 4-114 of SEQ ID NO: 12, and the operably linked reporter or nucleic acid encoding the TphR regulated promoter arrangement.

Across the independent claims, the core inventive scope is anchored by a TPA responsive regulatory system using a Comamonas testosteroni (TphR) transcription factor, with a TphR regulated promoter specifically defined by nucleotides 4-114 of SEQ ID NO: 12. The first independent claim further specifies an operably linked reporter protein for biosensor readout, while the second independent claim is directed to a nucleic acid defined by that same subsequence and encoding a TphR regulated promoter arrangement.

Stated Advantages

Documented Applications