2.1.1 Transcription factor activation/repression
A transcription factor (TF) is a protein molecule with a specific structure that binds to a specific gene and regulates gene expression at a specific intensity at a specific time and place. Therefore, the sensitivity and specificity of TFs can be used to design the sensor as a recognition element. TF-based cell-free biosensors have been used to identify specific small molecules, such as inorganic ions and organic molecules (bacterial quorum sensing molecules) [19], and they are also used in high-throughput screening and metabolic engineering [20]. Specific correct TFs or natural conformational TFs are selected by detecting different analytes, such as TF in response to aromatic compounds (XylS-AraC,XylR-NtrC, and LysR), metal ions (MerR, ArsR, DtxR, Fur, and NikR), or antibiotics (TetR and MarR) [21]. When analytes (ligands) are present in a cell-free environment, the response of TFs is immediately obtained, and binding of ligands to their activated TFs or the release of ligand-bound inhibitory TFs can lead to the reporter gene expression (Fig. 2A). For unknown ligands, they can be converted into detectable ligands by using metabolic enzymes [12].
To avoid the reaction of TFs to substances other than detection targets, TFs with enhanced specificity and sensitivity should be further expanded. Alternatively, a directed evolutionary strategy can be used to screen and select specific TFs [22]. Due to the known limitations of TFs, not all analytes can be identified, so there are other identification mechanisms [23, 24].