Improved Cell-Free Transcription-Translation Reactions in Microfluidic Chemostats Augmented with Hydrogel Membranes for Continuous Small Molecule Dialysis.

Increasing the protein production capacity of the PURE cell-free transcription-translation (TX-TL) system will be key to implementing complex synthetic biological circuits, and to establishing a fully self-regenerating system as a basis for the development of a synthetic cell. Under steady-state conditions, the protein synthesis capacity of the PURE system is likely at least one order of magnitude too low to express sufficient quantities of all PURE protein components. This is in part due to the fact that protein synthesis cannot be sustained during the entire dilution cycle, especially at low dilution rates. We developed a microfluidic chemostat augmented with semipermeable membranes that combines steady-state reactions and continuous dialysis as a possible solution to enhance protein synthesis at steady-state. In batch operation, the continuous dialysis of low molecular weight components via the membranes extended protein synthesis by over an order of magnitude from 2 h to over 30 h, leading to a 7-fold increase in protein yield. In chemostat operation, continuous dialysis enabled sustained protein synthesis during the entire dilution cycle even for low dilution rates, leading to 6-fold higher protein levels at steady state. The possibility to combine and independently manipulate continuous dialysis and chemostat operation renders our dialysis chemostat a promising technological basis for complex cell-free synthetic biology applications that require enhanced protein synthesis capacity.

Recombinase polymerase amplification in minimally buffered conditions.

Application of recombinase polymerase amplification (RPA) for pH-based detection of DNA amplification has been investigated. Commercial RPA kits from TwistDx are modified to minimize their pH buffering capacity. Due to the RPA's unique biochemistry, removal of tris from the amplification kit is not enough to lower the buffering capacity of the RPA assay. Even in the absence of tris, RPA components in the commercial kit intrinsically buffer the pH. We show different strategies to minimize the buffering capacity of the RPA kit, while maintaining the amplification efficiency. Even in minimally buffered conditions, it is noticed that RPA's amplification yield is not high enough to overcome the assay's intrinsic buffering capacity. The effect of pyrophosphate precipitation in RPA on the reaction's pH have also been addressed. In conclusion, this work highlights strategies and considerations for the development of pH-based assays from nucleic acid amplification methods which involve ancillary enzymes that catalyze nucleotide hydrolysis.

Degradation study on molecules released from laser-based jet injector.

Development of needle-free methods to administer injectable therapeutics has been researched for a few decades. We focused our attention on a laser-based jet injection technique where the liquid-jet actuation mechanism is based on optical cavitation. This study investigates the potential damage to therapeutic molecules which are exposed to nanosecond laser pulses in the configuration of a compact laser-based jet injection device. Implementation of a pulsed laser source at 1574 nm wavelength allowed us to generate jets from pure water solutions and circumvent the need to reformulate therapeutics with absorbing dyes. We performed H1-NMR analysis on exposed samples of Lidocaine and δ-Aminolevulinic acid. We made several tests with linear and plasmid DNA to assess the structural integrity and functional potency after ejection with our device. The tests showed no significant degradation or detectable side products, which is promising for further development and eventually clinical applications.

Steady-State Cell-Free Gene Expression with Microfluidic Chemostats.

Cell-free synthetic biology offers an approach to building and testing gene circuits in a simplified environment free from the complexity of a living cell. Recent advances in microfluidic devices allowed cell-free reactions to run under nonequilibrium, steady-state conditions enabling the implementation of dynamic gene regulatory circuits in vitro. In this chapter, we present a detailed protocol to fabricate a microfluidic chemostat device which enables such an operation, detailing essential steps in photolithography, soft lithography, and hardware setup.

OnePot PURE Cell-Free System.

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic biology. Unfortunately, commercially available systems are costly, and their tunability is limited. In comparison, a home-made approach can be customized based on user needs. However, the preparation of home-made systems is time-consuming and arduous due to the need for ribosomes as well as 36 medium scale protein purifications. Streamlining protein purification by coculturing and co-purification allows for minimizing time and labor requirements. Here, we present an easy, adjustable, time- and cost-effective method to produce all PURE system components within 1 week, using standard laboratory equipment. Moreover, the performance of the OnePot PURE is comparable to commercially available systems. The OnePot PURE preparation method expands the accessibility of the PURE system to more laboratories due to its simplicity and cost-effectiveness.

A partially self-regenerating synthetic cell.

Self-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology.

Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner.

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 µg/mL at a cost of 0.09 USD/µL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.