Ramirez, C. C. | Search bioRxiv

bioRxiv2026

CADGE 2.0, Transcription-Translation-Coupled DNA Replication is Improved in a Chemically Modified Cell-Free System

In vitro directed evolution in synthetic microcompartments can generally support the evolution of genes with functions beyond affinity. The main challenge in the implementation of this strategy is the need to incorporate no more than a single DNA template molecule per microcompartment, thereby establishing a robust genotype-phenotype linkage, but which results in slow, inconsistent in vitro transcription and translation (IVTT) and poor DNA recovery after selection or screening. To address this challenge, we previously developed CADGE (Clonal Amplification-enhanceD Gene Expression) a strategy that allows the clonal amplification of linear gene-encoding DNA and coupled, in situ transcription-translation of the gene of interest. Here, we show that clonal amplification is highly sensitive to the cell-free systems composition and that robust, highly efficient cell-free DNA amplification via the CADGE platform can be achieved by replacing standard vendor-supplied energy mixes with DNA replication-optimized, homemade counterparts.

synthetic biology↗

bioRxiv2021

Fatty Alcohols, a Minor Component of the Tree Tobacco Surface Wax, Reduce Insect Herbivory

Despite decades of research resulting in a comprehensive understanding of epicuticular wax biosynthesis and metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. To develop a better understanding of this role, we investigated plant-herbivore interactions in four Nicotiana glauca (tree tobacco) genome edited mutants. This included [eceriferum1 (cer1), eceriferum3 (cer3), {beta}-ketoacyl-coA synthase6 (kcs6), and fatty acyl-coA reductase (far)] displaying a wide range of alkane and fatty alcohol abundances. Three interaction classes were examined: chewing herbivory with seven caterpillar and one snail species, phloem feeding with Myzus persicae (green peach aphid), and egg laying with Bemisia tabaci (sweet potato whitefly). We found that high wax load and alkane abundance did not reduce caterpillar or snail herbivory. However, fatty alcohol content was negatively correlated with caterpillar growth, suggesting a role in reducing insect herbivory despite its lower levels. Aphid reproduction and feeding activity were not correlated with wax load and composition but are potentially affected by altered cutin composition of cer1 mutants. When examining non-feeding activities, wax crystal morphology could explain the preference of B. tabaci to lay eggs on wildtype plants relative to cer1 and far mutants. Accordingly, the fatty alcohol wax component reduces caterpillar herbivory on the chemical level, but oviposition is increased when wax crystals are dense. The results suggest that this varied response between herbivore classes and species, at times displaying increased and at times reduced fitness in response to altered wax composition is in part a consequence of co-evolution that shaped the specific effects of different N. glauca metabolites such as anabasine and fatty alcohols in plant-herbivore interactions.

plant biology↗