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Gene regulation based on regulatory RNA is an important mechanism in cells and is increasingly used for regulatory circuits in synthetic biology. Toehold switches are rationally designed post-transcriptional riboregulators placed in the 5’ untranslated region of mRNA molecules. In the inactive state of a toehold switch, the ribosome-binding site is inaccessible for the ribosome. In the presence of a trigger RNA molecule protein production is turned on. Using antisense RNA against trigger molecules (anti-trigger RNA), gene expression can also be switched off again. We here study the utility and regulatory effect of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and anti-trigger transcription, whereas their cognate toehold switch, regulating expression of a reporter protein, is transcribed from a constitutive promoter. We explore various design options for the arrangement of the promoters and demonstrate that the resulting dynamic behavior is strongly influenced by transcriptional interference (TI) effects, leading to more than four-fold differences in expression levels. Our experimental results are consistent with previous findings that enhanced local RNA polymerase concentrations due to active promoters in close proximity lead to an increase in transcriptional activity of the strongest promoter in the circuits. Based on this insight, we selected optimum promoter designs and arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers and two anti-triggers that function as a post-transcriptional RNA regulatory exclusive OR (XOR) gate.

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far only few viable approaches are available for the printing of gels hosting liveEscherichia colibacteria. Here we develop a gentle bioprinting method based on an alginate/agarose bioink that enables precise printing ofE.coliinto three-dimensional hydrogel structures up to 10 mm in height. Addition of a calcium peroxide-based oxygen generation system enables maturation of fluorescent proteins deep within the printed structures. We utilize spatial patterning with the bioprinter to control different types of chemical interaction between bacteria. We first show quorum sensing-based chemical communication between engineered sender and receiver bacteria placed at different positions inside the bioprint, and then demonstrate the fabrication of barrier structures defined by non-motile bacteria that can guide the movement of chemotactic bacteria inside a gel.

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell‐free gene expression systems. One of the crucial steps during the preparation of cell extract‐based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell‐free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108 PFU/mL. State‐of‐the‐art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme‐assisted sonication (LAS) extracts, but we observed an increase in the release of DNA‐binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract., E. coli extracts can be prepared using a simple combination of lysozyme incubation and ultrasonication. We evaluate their performance in cell‐free protein expression via the expression of reporter proteins, the assembly of bacteriophages, and using proteomics. Our results show a synergistic effect of both lysis methods.

Interlocked molecules such as catenanes and rotaxanes, connected only via mechanical bonds have the ability to perform large-scale sliding and rotational movements, making them attractive components for the construction of artificial molecular machines and motors. We here demonstrate the realization of large, rigid rotaxane structures composed of DNA origami subunits. The structures can be easily modified to carry a molecular cargo or nanoparticles. By using multiple axle modules, rotaxane constructs are realized with axle lengths of up to 355 nm and a fuel/anti-fuel mechanism is employed to switch the rotaxanes between a mobile and a fixed state. We also create extended pseudo-rotaxanes, in which origami rings can slide along supramolecular DNA filaments over several hundreds of nanometres. The rings can be actively moved and tracked using atomic force microscopy., Rotaxanes are interlocked molecules that can undergo sliding and rotational movements and can be used in artificial molecular machines and motors. Here, Simmel and co-workers show a rigid rotaxane structures consisting of DNA origami subunits that can slide over several hundreds of nanometres.

Cell-free systems allow interference with gene expression processes without requiring elaborate genetic engineering procedures. This makes it ideally suited for rapid prototyping of synthetic biological parts. Inspired by nature's strategies for the control of gene expression via short antisense RNA molecules, we here investigated the use of small DNA (sDNA) for translational inhibition in the context of cell-free protein expression. We designed sDNA molecules to be complementary to the ribosome binding site (RBS) and the downstream coding sequence of targeted mRNA molecules. Depending on sDNA concentration and the promoter used for transcription of the mRNA, this resulted in a reduction of gene expression of targeted genes by up to 50-fold. We applied the cell-free sDNA technique (CF-sDNA) to modulate cell-free gene expression from the native T7 phage genome by suppressing the production of the major capsid protein of the phage. This resulted in a reduced phage titer, but at the same time drastically improved cell-free replication of the phage genome, which we utilized to amplify the T7 genome by more than 15 000-fold in a droplet-based serial dilution experiment. Our simple antisense sDNA approach extends the possibilities to exert translational control in cell-free expression systems, which should prove useful for cell-free prototyping of native phage genomes and also cell-free phage manipulation.

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far, only a few viable approaches are available for the printing of gels hosting live