A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria

Abstract

A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.

Genetic Parts and Enabling Tools for Biocircuit Design

“…Furthermore, the application of biocircuits will benefit from expanding the part categories portable across different species, especially the nonmodel bacteria with high clinical and industrial values. Examples of such parts include T7 RNA polymerase, CRISPRi system, and STAR . Finally, opportunities for discovering new parts exist in unexplored bioinformatic data and biological processes of nonmodel viruses and bacteria .…”

“…Examples of such parts include T7 RNA polymerase, 146 CRISPRi system, 147 and STAR. 91 Finally, opportunities for discovering new parts exist in unexplored bioinformatic data and biological processes of nonmodel viruses and bacteria. 148 Many parts derived from virus systems can function orthogonally to cellular regulation, thus showing more predictable behavior.…”

“…This system could modulate the timing and dynamics of other devices (e.g., pulse generator 89 and switchable feedback promoters 90 ) and be recapitulated in six Gram-negative bacterial strains. 91 CRISPR Systems. Originally part of a bacterial "immune system" against bacteriophages, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated proteins (Cas) became popular in genetic engineering due to the ability to target and cleave arbitrary nucleotide sequences.…”

“…STARs represent an enticing alternative to protein regulators, as they can be coupled to reporter RNA aptamers such as Spinach , and Broccoli to build regulation networks without translation. This system could modulate the timing and dynamics of other devices (e.g., pulse generator and switchable feedback promoters) and be recapitulated in six Gram-negative bacterial strains …”

Toward predictable universal genetic circuit design

“…T7 RNAP has been crucial in driving gene expression in universal circuits due to its excellent orthogonality and robustness against diverse host backgrounds, including gram‐negative and positive bacteria and eukaryotes. To further leverage these advantages for multiplexed power supply, the transferability of orthogonal T7 RNAP variants, σ fragments, and other phage‐derived RNAPs like SP6 RNAP [12] should be evaluated. Integrating other components in the orthogonal central dogma [13], such as DNA polymerases, ribosomes, and tRNAs, into the power supply module will further isolate the heterologous genetic circuits from undesired interactions with host components (Figure 1B).…”