GROQ-seq Platform Expansion: Design of growth-coupled measurements of T7 RNA Polymerase

T7 RNA polymerase (T7 RNAP) is a cornerstone of synthetic biology and is widely used for high-specificity transcription. However, despite its widespread use, predicting how its sequence variations influence function remains challenging, limiting its full potential in bioengineering applications. We propose to apply growth-based assays to systematically investigate the sequence-function relationship of T7 RNAP and develop predictive models. We will onboard a T7 RNAP circuit to the newly named growth-based quantitative sequencing (GROQ-seq) platform using a set of calibration variants that span the desired dynamic range. We will use this assay to study the relationship between polymerase sequence and promoter specificity, generating a dataset that will enable precise protein engineering for synthetic biology applications such as mRNA therapeutics and biosensors.