A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

Abstract

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within five minutes after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase

“…To do this, we developed a transposase-based method that uses a novel transposon to split proteins, which we refer to as a ‘splitposon’. Previous methods have been published to generate libraries of split proteins or domain insertions that are based on incremental truncation (Ostermeier et al , 1999 ; Paschon & Ostermeier, 2004 ), multiplex inverse PCR (Kanwar et al , 2013 ), DNAse cleavage (Guntas & Ostermeier, 2004 ; Chen et al , 2009 ), and transposon insertion (Segall-Shapiro et al , 2011 ; Mahdavi et al , 2013 ). The transposon-based approaches are able to generate large libraries and do not require sensitive DNAse steps, but they leave ∼10 added amino acids at the split site.…”

Chemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics

“…bisected the NLL-EcMetRS and found several split variants capable of charging Anl to tRNA Met . 48 Because labeling requires expression of both the N- and C-terminal fragments of the synthetase, this system allows the investigator to restrict labeling to cells in which two promoters of interest are active.…”

Transplantation of prokaryotic two-component signaling pathways into mammalian cells

“…The experiments above show that the response requires expression of both pathway components. This can be described as AND logic (12,(41)(42)(43) between HK and RR genes as inputs. Controlling these genes with external stimuli can generate additional logic behaviors.…”