Abstract
Cell-free transcription−translation (TXTL) systems expressing genes from linear dsDNA enable the rapidprototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressiblecost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systemsthat reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bpGFP10−11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with itscomplementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used tocharacterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements.We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize ourconcept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction affordedby split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptideproduction context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. Weanticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interactionassays.