PlasmidMaker: a Versatile, Automated, and High Throughput End-to-End Platform for Plasmid Construction

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high-throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments within 3 days. PlasmidMaker should greatly expand the potential of synthetic biology.

Spatial constraints also regulates final achene mass in the sunflower (Helianthus annuus L.) capitulum

In capitula of the cultivated sunflower (<em>Helianthus annuus</em> L.) achene size and mass commonly decrease from proximal to distal positions. Temporal limitation of resources of the distal achenes over the proximal ones has been the common explanation for this response. Nevertheless, because the capitulum architecture and expansion dynamics also interacts with achene growth and development, also space exert a coupled effect with resources on achene size along the inflorescence radius. In this work we removed young achenes from different capitulum positions [inner sector (IS) and outer sector (OS)] and applied an artificial restriction to the capitulum/achenes radial expansion. Removal of outer achenes significantly increased the final dry mass of the remnant ones between 17.1 to 27.6%. Removal of inner achenes also produced the same effect but in less magnitude, between 9.3 to 17.9% of the outer ones. The removal of outer achenes with the application of an artificial peripheral constraint did not significantly increase the dry mass of the remnant ones (2.7% of the inner and 7.1% of the control). Percentage of empty achenes significantly diminished in the middle sector (MS) in capitula with the outer achenes removed and in capitula with the outer achenes removed plus a peripheral constraint but in the range of 7.1% (MS achenes) and 2.7 % (IS achenes). Percentage of empty achenes of the MS did not change when the outer achenes were removed but was significantly lower when the OS was removed and the peripheral constraint was applied. This results suggest that a part of the reduced growth and development of IS and MS achenes is not only controlled by the competition for resources but also is restricted by space and pressure exerted by the neighboring ones.