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Abstract Mars is a particularly attractive candidate among known astronomical objects to potentially host life. Results from space exploration missions have provided insights into Martian geochemistry that indicate oxychlorine species, particularly perchlorate, are ubiquitous features of the Martian geochemical landscape. Perchlorate presents potential obstacles for known forms of life due to its toxicity. However, it can also provide potential benefits, such as producing brines by deliquescence, like those thought to exist on present-day Mars. Here we show perchlorate brines support folding and catalysis of functional RNAs, while inactivating representative protein enzymes. Additionally, we show perchlorate and other oxychlorine species enable ribozyme functions, including homeostasis-like regulatory behavior and ribozyme-catalyzed chlorination of organic molecules. We suggest nucleic acids are uniquely well-suited to hypersaline Martian environments. Furthermore, Martian near- or subsurface oxychlorine brines, and brines found in potential lifeforms, could provide a unique niche for biomolecular evolution.

Abstract Biological computation is becoming a viable and fast-growing alternative to traditional electronic computing. Here we present a biocomputing technology called Trumpet: Transcriptional RNA Universal Multi-Purpose GatE PlaTform. Trumpet combines the simplicity and robustness of the simplest in vitro biocomputing methods, adding signal amplification and programmability, while avoiding common shortcomings of live cell-based biocomputing solutions. We have demonstrated the use of Trumpet to build all universal Boolean logic gates. We have also built a web-based platform for designing Trumpet gates and created a primitive processor by networking several gates as a proof-of-principle for future development. The Trumpet offers a change of paradigm in biocomputing, providing an efficient and easily programmable biological logic gate operating system.

Abstract Background Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Abstract Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.

Ions in the Hofmeister series exhibit varied effects on biopolymers. Those classed as kosmotropes generally stabilize secondary structure, and those classed as chaotropes generally destabilize secondary structure. Here, we report that several anionic chaotropes exhibit unique effects on one DNA secondary structure - a G quadruplex. These chaotropes exhibit the expected behaviour (destabilization of secondary structure) in two other structural contexts: a DNA duplex and i-Motifs. Uniquely among secondary structures, we observe that G quadruplexes are comparatively insensitive to the presence of anionic chaotropes, but not other denaturants. Further, the presence of equimolar NaCl provided greater mitigation of the destabilization caused by other non-anionic denaturants. These results are consistent with the presence of monovalent cations providing an especially pronounced stabilizing effect to G quadruplexes when studied in denaturing solution conditions.

Early cells likely consisted of fatty acid vesicles enclosing magnesium-dependent ribozymes. Here, the authors show that fatty acid derivatives can form vesicles that, unlike those formed from only unmodified fatty acids, are stable in the presence of magnesium and could support ribozyme catalysis.

DNA oligonucleotides containing 8-aminodeoxyguanosine residues form a pH-dependent self-structure with strong hydrogen bonds. [ABSTRACT FROM AUTHOR]

Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.