Modular Golden Gate Assembly of Linear DNA Templates for Cell-Free Prototyping.

Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox.

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity.

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions. Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown.

RNA contact prediction by data efficient deep learning.

On the path to full understanding of the structure-function relationship or even design of RNA, structure prediction would offer an intriguing complement to experimental efforts. Any deep learning on RNA structure, however, is hampered by the sparsity of labeled training data. Utilizing the limited data available, we here focus on predicting spatial adjacencies ("contact maps") as a proxy for 3D structure.

Microfluidic Production of Porous Polymer Cell-Mimics Capable of Gene Expression.

Engineering simple, artificial models of living cells allows synthetic biologists to study cellular functions under well-controlled conditions. Reconstituting multicellular behaviors with synthetic cell-mimics is still a challenge because it requires efficient communication between individual compartments in large populations. This chapter presents a microfluidic method to produce large quantities of cell-mimics with highly porous, stable, and chemically modifiable polymer membranes that can be programmed on demand with nucleus-like DNA-hydrogel compartments for gene expression.

Functionalizing Cell-Free Systems with CRISPR-Associated Proteins: Application to RNA-Based Circuit Engineering.

Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far, the production of cell-free extracts has often been decoupled from the production of functionalized proteins.