Biology subjects

Maerkl, S. J.

Publications and source records attributed to Maerkl, S. J..

6 recordsLinked to original sources

bioRxiv2018

A simple, robust, and low-cost method to produce the PURE cell-free system

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 {micro}g/mL at a cost of 0.09 USD/{micro}L, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The OnePot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

synthetic biology↗

bioRxiv2018

Cell-free gene regulatory network engineering with synthetic transcription factors

Gene regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function that can be tuned at the level of DNA, protein, and cooperative protein - protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription factor binding energy landscapes and mechanistic modeling, enabling us to generate a library of well-characterized synthetic transcription factors and corresponding promoters, which we then used to build gene regulatory networks de novo. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene regulatory networks and for studying the biophysics of transcriptional regulation.

synthetic biology↗

bioRxiv2018

Microfluidic device for on-chip mixing and encapsulation of lysates

Emulsion drops are often employed as picoliter-sized containers to perform screening assays. These assays usually entail the formation of drops encompassing discrete objects such as cells or microparticles and reagents to study interactions between the different encapsulants. Drops are also used to screen influences of reagent concentrations on the final product. However, these latter assays are less frequently performed because it is difficult to change the reagent concentration over a wide range with high precision within a single experiment. In this paper, we present a microfluidic double emulsion drop maker containing pneumatic valves that enable injection of different reagents using pulsed width modulation and subsequent mixing. This device can produce drops from reagent volumes as low as 10 l with minimal sample loss, thereby enabling experiments that would be prohibitively expensive using droplet generators that do not contain valves. We employ this device to monitor the kinetics of cell free synthesis of green fluorescent proteins inside double emulsions. To demonstrate the potential of this device, we perform DNA titration experiments in double emulsion drops to test the influence of the DNA concentration on the amount of green fluorescence proteins produced.

synthetic biology↗

bioRxiv2017

Microfluidic Transfection for High-Throughput Mammalian Protein Expression

Mammalian synthetic biology and cell biology would greatly benefit from improved methods for highly parallel transfection, culturing and interrogation of mammalian cells. Transfection is routinely performed on high-throughput microarrays, but this setup requires manual cell culturing and precludes precise control over the cell environment. As an alternative, microfluidic transfection devices streamline cell loading and culturing. Up to 280 transfections can be implemented on the chip at high efficiency. The culturing environment is tightly regulated and chambers physically separate the transfection reactions, preventing cross-contamination. Unlike typical biological assays that rely on end-point measurements, the microfluidic chip can be integrated with high-content imaging, enabling the evaluation of cellular behavior and protein expression dynamics over time.

molecular biology↗

bioRxiv2017

A Microfluidic Module For Real-Time Generation Of Complex Multi-Molecule Temporal Concentration Profiles

We designed a microfluidic module that generates complex, dynamic concentration profiles of multiple molecules over a large concentration range using pulse-width modulation (PWM). Our PWM device can arbitrarily combine up to 6 different inputs and select between three downstream mixing channels as required by the application. The module can produce arbitrary concentrations with a dynamic range of up to 3-5 decades. We created complex concentration profiles of 2 molecules, with each concentration independently controllable, and show that the PWM module can execute rapid concentration changes as well as long-timescale pharmacokinetic profiles. Concentration profiles were generated for molecules with molecular weights ranging from 560 Da to 150 kDa. Our PWM module produces robust and precise concentration profiles under a variety of operating conditions, making it ideal for integration with existing microfluidic devices for advanced cell and pharmacokinetic studies.

bioengineering↗

bioRxiv2017

A microfluidic biodisplay

Synthetically engineered cells are powerful and potentially useful biosensors, but it remains problematic to deploy such systems due to practical difficulties and biosafety concerns. To overcome these hurdles, we developed a microfluidic device that serves as an interface between an engineered cellular system, environment, and user. We created a biodisplay consisting of 768 individually programmable biopixels and demonstrated that it can perform multiplexed, continuous sampling. The biodisplay detected 10 {micro}g/l sodium-arsenite in tap water using a research grade fluorescent microscope, and reported arsenic contamination down to 20 {micro}g/l with an easy to interpret \"skull and crossbones\" symbol detectable with a low-cost USB microscope or by eye. The biodisplay was designed to prevent release of chemical or biological material to avoid environmental contamination. The microfluidic biodisplay thus provides a practical solution for the deployment and application of engineered cellular systems.

synthetic biology↗