Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants

TLDR

Synthetic gene circuits require orthogonal regulatory parts, yet the limited number of well‑characterized transcription factors hampers scaling and complexity. The authors engineered a library of transcriptional AND gates by splitting T7 RNA polymerase and mutating the C‑terminal DNA‑recognition domain to alter promoter specificity. They validated that split T7 RNAP is active in vivo, compared it to the full‑length enzyme, and constructed a collection of mutant split RNAPs with diverse activities paired with corresponding altered promoters. Assays of wild‑type and mutant split RNAPs demonstrated that regulated expression of the N‑ and C‑terminal fragments produces robust AND logic, establishing mutant split T7 RNAP as a versatile component for synthetic gene circuits.

Abstract

The construction of synthetic gene circuits relies on our ability to engineer regulatory architectures that are orthogonal to the host’s native regulatory pathways. However, as synthetic gene circuits become larger and more complicated, we are limited by the small number of parts, especially transcription factors, that work well in the context of the circuit. The current repertoire of transcription factors consists of a limited selection of activators and repressors, making the implementation of transcriptional logic a complicated and component-intensive process. To address this, we modified bacteriophage T7 RNA polymerase (T7 RNAP) to create a library of transcriptional AND gates for use in Escherichia coli by first splitting the protein and then mutating the DNA recognition domain of the C-terminal fragment to alter its promoter specificity. We first demonstrate that split T7 RNAP is active in vivo and compare it with full-length enzyme. We then create a library of mutant split T7 RNAPs that have a range of activities when used in combination with a complimentary set of altered T7-specific promoters. Finally, we assay the two-input function of both wild-type and mutant split T7 RNAPs and find that regulated expression of the N- and C-terminal fragments of the split T7 RNAPs creates AND logic in each case. This work demonstrates that mutant split T7 RNAP can be used as a transcriptional AND gate and introduces a unique library of components for use in synthetic gene circuits.