Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of l-lysine by l-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational l-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinked.

Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane.

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

Protocol to identify amino acids bound to tRNA by aminoacylation using mass spectrometry.

tRNA-bound amino acids often need to be identified, for instance, in cases where different amino acids compete for binding to the same tRNA. Here, we present a mass-spectrometry-based protocol to determine the amino acids bound to tRNA by aminoacylation. We detail how to perform the aminoacylation reaction, the preparation of the aminoacyl-tRNA for measurement, and the mass spectrometric analysis. We use arginine acylation as an example; however, this protocol can be applied to any other amino acid.

The Nature of the Spark Is a Pivotal Element in the Design of a Miller-Urey Experiment.

Miller and Urey applied electric sparks to a reducive mixture of CH4, NH3, and water to obtain a complex organic mixture including biomolecules. In this study, we examined the impact of temperature, initial pressure, ammonia concentration, and the spark generator on the chemical profile of a Miller-Urey-type prebiotic broth. We analyzed the broth composition using Gas Chromatography combined with Mass Spectroscopy (GC/MS). The results point towards strong compositional changes with the nature of the spark. Ammonia exhibited catalytic properties even with non-nitrogen-containing compounds. A more elevated temperature led to a higher variety of substances. We conclude that to reproduce such a broth as well as possible, all the studied parameters need to be tightly controlled, the most difficult and important being spark generation.

A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA Reconstituted in an All E. coli Cell-Free Expression System.

Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems.

Cell-free transcription-translation systems are a versatile tool to study gene expression, enzymatic reactions and biochemical regulation mechanisms. Because cell-free transcription-translation systems are often derived from cell lysates, many different substances, among them amino acids, are present. However, experiments concerning the incorporation of non-canonical amino acids into proteins require a system with negligible amounts of canonical analogs. Here we propose a two-step method for the removal of residual free lysine in an all Escherichia coli-based cell-free expression system. The first step consists of the expression of a high-lysine dummy protein. The second step consists of direct removal via binding between lysine and DNA. The presented method is an efficient, fast and simple way to remove residual lysine without altering the system ability to perform gene expression.

Bead-based assay for spatiotemporal gene expression control in cell-free transcription-translation systems.

Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a bead-based method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.