Optimisation of TP53 reporters by systematic dissection of synthetic TP53 response elements
In this project, we systematically designed >1,200 barcoded TP53 reporters to probe the impact of TP53 binding site (BS) affinity, BS spacing, spacer sequence identity, and core promoter on transcriptional activity. The reporter pool was transfected into TP53 proficient MCF7, A549 and U2OS cells or into polyclonal TP53-knockout MCF7 cells. Additionally, the cells were stimulated using Nutlin-3a. The barcodes in the RNA were then synthesised to DNA and measured by high-throughput sequencing. Reporter activities were computed by comparing the barcode counts in the cDNA to the barcode counts in the input plasmid DNA. We found that the design of the TP53 reporter substantially alters the transcriptional output.