Biology subjects

Park, D. M.

Publications and source records attributed to Park, D. M..

4 recordsLinked to original sources

bioRxiv2023

GENTANGLE: integrated computational design of gene entanglements

SummaryThe design of two overlapping genes in a microbial genome is an emerging technique for adding more reliable control mechanisms in engineered organisms for increased safety. The design of functional gene pairs is a challenging procedure and computational design tools are used to improve the efficiency to deploy successful designs in genetically engineered systems. GENTANGLE (Gene Tuples ArraNGed in overLapping Elements) is a high performance containerized pipeline for the computational design of two overlapping genes translated in different reading frames of the genome. This new software package can be used to design and test gene entanglements for microbial engineering projects using arbitrary sets of user specified gene pairs. Availability and ImplementationThe GENTANGLE source code and its submodules are freely available on GitHub at https://github.com/BiosecSFA/gentangle. The DATANGLE (DATA for genTANGLE) repository contains related data and results, and is freely available on GitHub at https://github.com/BiosecSFA/datangle. The GENTANGLE repository wiki contains detailed instructions on how to use the container and the different components of software and data, including reproducing the results. The code is licensed under the GNU Affero General Public License version 3 (https://www.gnu.org/licenses/agpl.html). Contactmartimartine1@llnl.gov and allen99@llnl.gov

bioinformatics↗

bioRxiv2023

Scalable bio-platform to recover critical metals fromcomplex waste sources

Chemical methods for extraction and refinement of technologically critical rare earth elements (REEs) are energy intensive, hazardous, and environmentally destructive. Current bio-based extraction systems rely on extremophilic organisms and generate many of the same detrimental effects as chemical methodologies. The mesophilic methylotrophic bacterium Methylobacterium extorquens AM1 was previously shown to grow using electronic waste by naturally acquiring REEs to power methanol metabolism. Here we show that growth using electronic waste as a sole REE source is scalable up to 10 L with consistent metal yields, without the use of harsh acids or high temperatures. Addition of organic acids increases REE leaching in a nonspecific manner. REE-specific bioleaching can be engineered through the overproduction of REE-binding ligands (called lanthanophores) and pyrroloquinoline quinone. REE bioaccumulation increases with leachate concentration and is highly specific. REEs are stored intracellularly in polyphosphate granules, and genetic engineering to eliminate exopolyphosphatase activity increases metal accumulation, confirming the link between phosphate metabolism and biological REE use. Finally, we report the innate ability of M. extorquens to grow using other complex REE sources including pulverized smart phones, demonstrating the flexibility and potential for use as a recovery platform for these critical metals.

bioengineering↗

bioRxiv2023

Prolonging Genetic Circuit Stability through Adaptive Evolution of Overlapping Genes

The development of synthetic biological circuits that maintain functionality over application relevant timescales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or "entangled" entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for over 130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the mutational landscape such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more benign mutations lowered circuit burden, which suppressed the accumulation of relE inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

synthetic biology↗

bioRxiv2022

Comprehensive analysis of kill switch toxins in plant-beneficial Pseudomonas fluorescens reveals drivers of lethality, stability, and escape

Kill switches provide a biocontainment strategy in which unwanted growth of an engineered microorganism is prevented by expression of a toxin gene. A major challenge in kill switch engineering is balancing evolutionary stability with robust cell killing activity in application relevant host strains. Understanding host-specific containment dynamics and modes of failure helps to develop potent yet stable kill switches. To guide the design of robust kill switches in the agriculturally relevant strain Pseudomonas fluorescens SBW25, we present a comparison of lethality, stability, and genetic escape of eight different toxic effectors in the presence of their cognate inactivators (i.e., toxin-antitoxin modules, polymorphic exotoxin-immunity systems, restriction endonuclease-methyltransferase pair). We find that cell killing capacity and evolutionary stability are inversely correlated and dependent on the level of protection provided by the inactivator gene. Decreasing the proteolytic stability of the inactivator protein can increase cell killing capacity, but at the cost of long-term circuit stability. By comparing toxins within the same genetic context, we determine that modes of genetic escape increase with circuit complexity and are driven by toxin activity, the protective capacity of the inactivator, and the presence of mutation-prone sequences within the circuit. Collectively, our study reveals that circuit complexity, toxin choice, inactivator stability, and DNA sequence design are powerful drivers of kill switch stability and valuable targets for optimization of biocontainment systems.

synthetic biology↗