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].