Single 3'-exonuclease-based multifragment DNA assembly method (SENAX).

DNA assembly is a vital process in biotechnology and synthetic biology research, during which DNA plasmids are designed and constructed using bioparts to engineer microorganisms for a wide range of applications. Here, we present an enzymatic homology-based DNA assembly method, SENAX (Stellar ExoNuclease Assembly miX), that can efficiently assemble multiple DNA fragments at ambient temperature from 30 to 37 °C and requires homology overlap as short as 12-18 base pairs. SENAX relies only on a 3'-5' exonuclease, XthA (ExoIII), followed by Escherichia coli transformation, enabling easy scaling up and optimization. Importantly, SENAX can efficiently assemble short fragments down to 70 bp into a vector, overcoming a key shortcoming of existing commonly used homology-based technologies. To the best of our knowledge, this has not been reported elsewhere using homology-based methods. This advantage leads us to develop a framework to perform DNA assembly in a more modular manner using reusable promoter-RBS short fragments, simplifying the construction process and reducing the cost of DNA synthesis. This approach enables commonly used short bioparts (e.g., promoter, RBS, insulator, terminator) to be reused by the direct assembly of these parts into intermediate constructs. SENAX represents a novel accurate, highly efficient, and automation-friendly DNA assembly method.

Thermogenetics: Applications come of age.

Temperature is a ubiquitous physical cue that is non-invasive, penetrative and easy to apply. In the growing field of thermogenetics, through beneficial repurposing of natural thermosensing mechanisms, synthetic biology is bringing new opportunities to design and build robust temperature-sensitive (TS) sensors which forms a thermogenetic toolbox of well characterised biological parts. Recent advancements in technological platforms available have expedited the discovery of novel or de novo thermosensors which are increasingly deployed in many practical temperature-dependent biomedical, industrial and biosafety applications. In all, the review aims to convey both the exhilarating recent technological developments underlying the advancement of thermosensors and the exciting opportunities the nascent thermogenetic field holds for biomedical and biotechnology applications.

Highly Reversible Tunable Thermal-Repressible Split-T7 RNA Polymerases (Thermal-T7RNAPs) for Dynamic Gene Regulation.

Temperature is a physical cue that is easy to apply, allowing cellular behaviors to be controlled in a contactless and dynamic manner via heat-inducible/repressible systems. However, existing heat-repressible systems are limited in number, rely on thermal sensitive mRNA or transcription factors that function at low temperatures, lack tunability, suffer delays, and are overly complex. To provide an alternative mode of thermal regulation, we developed a library of compact, reversible, and tunable thermal-repressible split-T7 RNA polymerase systems (Thermal-T7RNAPs), which fused temperature-sensitive domains of Tlpa protein with split-T7RNAP to enable direct thermal control of the T7RNAP activity between 30 and 42 °C. We generated a large mutant library with varying thermal performances via an automated screening framework to extend temperature tunability. Lastly, using the mutants, novel thermal logic circuitry was implemented to regulate cell growth and achieve active thermal control of the cell proportions within co-cultures. Overall, this technology expanded avenues for thermal control in biotechnology applications.

Biodegradation of natural rubber and deproteinized natural rubber by enrichment bacterial consortia.

This study examined the biodegradation of natural rubber (NR) and deproteinized natural rubber (DPNR) by bacterial consortia enriched from a rubber-processing factory's waste in Vietnam. The results reveal the degradation in both NR and DPNR, and the DPNR was degraded easier than NR. The highest weight loss of 48.37% was obtained in the fourth enrichment consortium with DPNR, while 35.39% was obtained in the fifth enrichment consortium with NR after 14 days of incubation. Nitrogen content and fatty acid content determined by Kjeldahl method and fourier transform infrared spectroscopy (FTIR), respectively, were decreased significantly after being incubated with the consortia. Structure of degraded rubber film analyzed by nuclear magnetic resonance spectroscopy showed the presence of aldehyde group, a sign of rubber degradation. Bacterial cells tightly adhering and embedding into NR and DPNR films were observed by scanning electron microscopy. There were differences in the bacterial composition of the consortia with NR and DPNR, which were determined by metagenomic analysis using 16S rRNA gene sequencing. The phyla Bacteroidetes and Proteobacteria may play a role in the degradation of non-isoprene compounds such as protein or lipid, while the phylum Actinobacteria plays a crucial role in the degradation of rubber hydrocarbon in all consortia.