Biotechnology Applications of Cell-Free Expression Systems

Cell-free systems are a rapidly expanding platform technology with an important role in the engineering of biological systems. The key advantages that drive their broad adoption are increased efficiency, versatility, and low cost compared to in vivo systems. Traditionally, in vivo platforms have been used to synthesize novel and industrially relevant proteins and serve as a testbed for prototyping numerous biotechnologies such as genetic circuits and biosensors. Although in vivo platforms currently have many applications within biotechnology, they are hindered by time-constraining growth cycles, homeostatic considerations, and limited adaptability in production. Conversely, cell-free platforms are not hindered by constraints for supporting life and are therefore highly adaptable to a broad range of production and testing schemes. The advantages of cell-free platforms are being leveraged more commonly by the biotechnology community, and cell-free applications are expected to grow exponentially in the next decade. In this study, new and emerging applications of cell-free platforms, with a specific focus on cell-free protein synthesis (CFPS), will be examined. The current and near-future role of CFPS within metabolic engineering, prototyping, and biomanufacturing will be investigated as well as how the integration of machine learning is beneficial to these applications.

Regularities in the formation of entomofauna in alfalfa agrocenosis

The field work was performed at the experimental field of the Institute of Forage Crops, Pleven, Bulgaria during the period of 2006-2009. The share distribution of the orders Coleoptera, Thysanoptera, Diptera, Orthoptera and Hemiptera, suborder Sternorrhyncha, Heteroptera, Fulgoromorpha and Cicadomorpha was established for every four alfalfa growth cycles during the vegetation period for four years. It was found that the share participation of orders was determined primarily by the population dynamics of the dominant insect species and it was closely dependent on plant development following a characteristic course over the years. Depending on the food specialization and the division of the species into harmful and beneficial, the share of predators in regrowth corresponded to that of their prey. A clear trend was observed of increasing the share of harmful species and reducing the beneficial ones (Coleoptera, Thysanoptera, Hemiptera: Heteroptera) with the alfalfa development over the years.

QTL Map of Early- and Late-Stage Perennial Regrowth in Zea diploperennis

Numerous climate change threats will necessitate a shift toward more sustainable agricultural practices during the 21st century. Conversion of annual crops to perennials that are capable of regrowing over multiple yearly growth cycles could help to facilitate this transition. Perennials can capture greater amounts of carbon and access more water and soil nutrients compared to annuals. In principle it should be possible to identify genes that confer perenniality from wild relatives and transfer them into existing breeding lines to create novel perennial crops. Two major loci controlling perennial regrowth in the maize relative Zea diploperennis were previously mapped to chromosome 2 (reg1) and chromosome 7 (reg2). Here we extend this work by mapping perennial regrowth in segregating populations involving Z. diploperennis and the maize inbreds P39 and Hp301 using QTL-seq and traditional QTL mapping approaches. The results confirmed the existence of a major perennial regrowth QTL on chromosome 2 (reg1). Although we did not observe the reg2 QTL in these populations, we discovered a third QTL on chromosome 8 which we named regrowth3 (reg3). The reg3 locus exerts its strongest effect late in the regrowth cycle. Neither reg1 nor reg3 overlapped with tiller number QTL scored in the same population, suggesting specific roles in the perennial phenotype. Our data, along with prior work, indicate that perennial regrowth in maize is conferred by relatively few major QTL.

Rapid evolution of bacterial mutualism in the plant rhizosphere

While beneficial plant-microbe interactions are common in nature, direct evidence for the evolution of bacterial mutualism is scarce. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacteria evolve into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition is accompanied with increased mutualist fitness via two mechanisms: (i) improved competitiveness for root exudates and (ii) enhanced tolerance to the plant-secreted antimicrobial scopoletin whose production is regulated by transcription factor MYB72. Crucially, these mutualistic adaptations are coupled with reduced phytotoxicity, enhanced transcription of MYB72 in roots, and a positive effect on plant growth. Genetically, mutualism is associated with diverse mutations in the GacS/GacA two-component regulator system, which confers high fitness benefits only in the presence of plants. Together, our results show that rhizosphere bacteria can rapidly evolve along the parasitism-mutualism continuum at an agriculturally relevant evolutionary timescale.

Heterogeneous Growth Cycles

This paper focuses on the Dirichlet and the Dirichlet-multinomial distributions to analyze one definition of the cycle based on three economic performance regimens: augmentation, stagnation and diminution. We use data from 142 economies grouped in seven clusters during the second half of the last century, a period which has witnessed various structural changes. Furthermore, this paper advances to investigate what the probabilities are of reaching significant positive growth rates ⎯the frequency of the augmentation regimen⎯ which prevailed during the decade from 1950 to 1960, using Bayesian statistics.