A transcription activator–like effector (TALE) induction system mediated by proteolysis

Abstract

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. In this work we expand the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic.

Controlling localization of Escherichia coli populations using a two‐part synthetic motility circuit: An accelerator and brake

“…By placing cheZ under control of SoxR, our goal was to achieve pseudotaxis toward pyocyanin. Our design concept included active degradation of CheZ by the addition of the C‐terminal ClpXP‐targeting domain of YbaQ (Baker & Sauer, ; Flynn, Neher, Kim, Sauer, & Baker, ; Tschirhart et al, ) for enabling rapid turnover and better coordination of the transient CheZ level presumably relative to the CheY level, the result being streamlined regulation of the length and duration of the bacterial “runs.“ Programmed or tailored protein level‐degradation (McGinness, Baker, & Sauer, ) has worked in many systems of metabolic engineering and synthetic biology (Brockman & Prather, ; Copeland, Politz, Johnson, Markley, & Pfleger, ; Sekar, Gentile, Bostick, & Tyo, ), however, to our knowledge few have utilized this concept to help guide pseudotaxis (Hwang et al, ). If engineered pseudotactic cells migrate toward and into a gradient of the pseudoattractant, CheZ proteins will be produced to confer motility.…”

Evolution of a split RNA polymerase as a versatile biosensor platform

“…RNA is a particularly useful output due to the extensive technologies available to drive cellular responses based on computed nucleic acid signals 5–7 . Nature utilizes transcription factors to detect target activities and drive genetic responses, and many naturally occurring transcription factors, such as the Lac and Tet repressors, have been repurposed as gene expression control elements 4,8 . However, because the scope of detection of such natural systems is limited, several engineered alternative technologies have been developed.…”

“…The development of programmable DNA binding domains such as TALEs (Transcription activator-like effectors) and dCas9 have revolutionized 1- and 2-hybrid approaches 8,13–15 . However, a challenge with n-hybrids is that each system needs to be carefully tuned and optimized for each new interaction, and more complex multicomponent systems, such as 3-hybrids, often lack in sensitivity and signal-to-noise 16,17 .…”

Tobacco Etch Virus protease: A shortcut across biotechnologies

“…An inducible TEVp was recently used to modulate gene expression in E. coli (Copeland et al, 2016) targeting bacterial virulence factors called transcription activator-like effectors (TALEs).…”