A Versatile Toolbox for the Control of Protein Levels Using N?-Acetyl-Lysine Dependent Amber Suppression

In vivo analysis of essential genes often requires tight control of the levels of their protein products. Current methodologies focus mainly on transcriptional or post-transcriptional regulation of mRNAs. In the present study we developed a toolbox, based on amber suppression in the presence of N^ε-acetyl lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC to measure the effects of AcK-dependent amber suppression in Escherichia coli. We demonstrate here that, in combination with transcriptional regulation, the system allows for both total repression and gradual adjustment of protein production. To identify sequence motifs that provide improved translational regulation, we varied the sequence context of the amber codon and found that insertion of two preceding prolines significantly diminishes luminescence. In addition, using LacZ as a reporter, we demonstrated that a strain encoding a variant with a Pro-Pro amber motif can only grow on lactose when AcK is supplied, thus confirming the tight translational regulation of protein output. In parallel, we constructed an E. coli strain that carries an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible version of the gene for AcK-tRNA synthetase (AcKRS) on the chromosome, thus preventing mischarging of non-cognate substrates. Furthermore, we assembled a set of vectors based on the broad-host-range pBBR ori that enable the AcK-dependent amber suppression system to control protein output not only in E. coli, but also in Salmonella enterica and Vibrio cholerae.