Synthetic gene circuits, just like electronic circuits, are built from parts that take an input, process it, and pass on an output. The toolbox of parts for gene circuits has long been limited to those found somewhere in nature, particularly when it comes to sensing an input. Typically, these systems repurpose a natural transcription factor that senses the small molecule of interest. However, for the first time, a team from the California Institute of Technology have redesigned a transcription factor to respond to an entirely different small molecule.
The sensor of choice was qacR, a member of the TetR family of transcriptional repressors, which controls the expression of an efflux pump (qacA) in Staphylococcus aureus bacteria to remove antibiotics. The TetR repressor family responds to a broad range of antibiotics and has two domains; one is used to bind DNA and another to bind the ligand/effector (tetracycline or other antibiotics). When the effector isn’t present, qacR binds to regulatory elements to prevent the expression of qacA. However, once the effector arrives, it causes a conformational change in qacR that releases it from the DNA and relieves repression of downstream genes.
This system has been exploited for numerous synthetic circuits, but never before with an effector so different from what nature has intended. The target of choice in this case was vanillin, which, aside from it’s claim to culinary fame, is a growth-inhibiting small molecule that is a troublesome byproduct in the production of biofuel and other industrial processes.
Here’s how the team hacked qacR:
- They used computational protein design to analyze the known crystal structure of qacR and determine which combination of mutations would likely allow for the binding of such a different effector while still maintaining function, a critical library size-reducing step for downstream screening.
- After generating a library of sequences, they tested the response of the sensor using a cell-free transcription-translation (TX-TL) system:
- Cell-free TX-TL prototyping allows rapid prototyping and a test environment that is independent of cell wall permeability, toxicity, and interactions with a host cell.
- The cell-free system was first demonstrated using the wild-type protein and a GFP reporter downstream of the qacA promoter.
- They then tested mutants from their library against both the desired effector (vanillin) and the natural effector (the antibiotic dequalinium)
- Two mutants, differing by 11 and 6 mutations from the wild-type, showed a strong increase in GFP signal in response to vanillin and were functionally validated both in vitro and in vivo.
While functional, the redesigned sensors still had a few limitations, such as a reduced repression strength, which lowers the dynamic range compared to the wild-type protein, and some cellular toxicity that may be due to off-target effects. However, despite these prototype shortcomings, the team has shown for the first time that it is possible to design synthetic sensors to respond to a divergent effector of choice. The workflow of sequence selection aided by computational screens, screening with a cell-free TX-TL system, and then validating in vitro and in vivo is a valuable addition to the synthetic biologist’s toolkit. Furthermore, this novel protein can now be subjected to directed evolution to address the initial limitations and may one day end up as part of a system to deal with the problem of vanillin leaving a sour taste in the industrial biofuel process.
Go learn more about how to quickly design your own synthetic sensors in ACS Synthetic Biology, August 2015