Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

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.

Periodic Operation of a Dynamic DNA Origami Structure Utilizing the Hydrophilic-Hydrophobic Phase-Transition of Stimulus-Sensitive Polypeptides.

Dynamic DNA nanodevices are designed to perform structure-encoded motion actuated by a variety of different physicochemical stimuli. In this context, hybrid devices utilizing other components than DNA have the potential to considerably expand the library of functionalities. Here, the reversible reconfiguration of a DNA origami structure using the stimulus sensitivity of elastin-like polypeptides is reported. To this end, a rectangular sheet made using the DNA origami technique is functionalized with these peptides and by applying changes in salt concentration the hydrophilic-hydrophobic phase transition of these peptides actuate the folding of the structure. The on-demand and reversible switching of the rectangle is driven by externally imposed temperature oscillations and appears at specific transition temperatures. Using transmission electron microscopy, it is shown that the structure exhibits distinct conformational states with different occupation probabilities, which are dependent on structure-intrinsic parameters such as the local number and the arrangement of the peptides on the rectangle. It is also shown through ensemble fluorescence resonance energy transfer spectroscopy that the transition temperature and thus the thermodynamics of the rectangle-peptide system depends on the stimuli salt concentration and temperature, as well as on the intrinsic parameters.

Communication and computation by bacteria compartmentalized within microemulsion droplets.

Amphiphilic inducer molecules such as N-acyl-L-homoserine lactones (AHLs) or isopropyl-ß-D-thio-galactopyranoside (IPTG) can be utilized for the implementation of an artificial communication system between groups of E. coli bacteria encapsulated within water-in-oil microemulsion droplets. Using spatially extended arrays of microdroplets, we study the diffusion of both AHL and IPTG from inducer-filled reservoirs into bacteria-containing droplets, and also from droplets with AHL producing sender bacteria into neighboring droplets containing receiver cells. Computational modeling of gene expression dynamics within the droplets suggests a strongly reduced effective diffusion coefficient of the inducers, which markedly affects the spatial communication pattern in the neighborhood of the senders. Engineered bacteria that integrate AHL and IPTG signals with a synthetic AND gate gene circuit are shown to respond only in the presence of both types of sender droplets, which demonstrates the potential of the system for genetically programmed pattern formation and distributed computing.

Transcriptional Interference in Toehold Switch-Based RNA Circuits.

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 to the ribosome. In the presence of a trigger RNA molecule, protein production is turned on. Using antisense RNA against trigger molecules (antitrigger RNA), gene expression can also be switched off again. We here study the utility of antisense transcription in this context, which enables a particularly compact circuit design. Our circuits utilize two inducible promoters that separately regulate trigger and antitrigger transcription, whereas their cognate toehold switch, regulating the 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 influenced by transcriptional interference (TI) effects depending on the promoter distance. 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. We observed that the range of this effect is larger when the participating promoters are stronger. Based on this insight, we combined two promoter arrangements for the realization of a genetic circuit comprised of two toehold switches, two triggers, and two antitriggers that function as a two-input two-output logic gate.

Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach.

CRISPR interference (CRISPRi) using dCas9-sgRNA is a powerful tool for the exploration and manipulation of gene functions. Here we quantify the reversible switching of a central process of the bacterial cell cycle by CRISPRi and an antisense RNA mechanism. Reversible induction of filamentous growth in E. coli has been recently demonstrated by controlling the expression levels of the bacterial cell division proteins FtsZ/FtsA via CRISPRi. If FtsZ falls below a critical level, cells cannot divide. However, the cells remain metabolically active and continue with DNA replication. We surmised that this makes them amenable to an inducible antisense RNA strategy to counteract FtsZ inhibition. We show that both static and inducible thresholds can adjust the characteristics of the switching process. Combining bulk data with single cell measurements, we characterize the efficiency of the switching process. Successful restoration of division is found to occur faster in the presence of antisense sgRNAs than upon simple termination of CRISPRi induction.

Single Cell Analysis of a Bacterial Sender-Receiver System.

Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as 'bacterial sensors' for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their 'sending power' is determined.