Molecular cloning is a routine yet essential technique. Its efficiency relies on ligation and can be greatly improved when using a procedure known as Golden Gate Assembly (GGA). Essential to GGA are type IIS enzymes that have the unique property to cleave downstream their recognition sequence and thus generate any non-palindromic overhangs. Today, GGA benefits from new engineered enzymes with enhanced activity. Concomitantly, high throughput GAG assays (involving the simultaneous study of all overhangs at a time) have proposed optimal GGA substrates with high efficiencies and fidelities. Surprisingly, those assays show either no or unexpected correlation between ligation efficiencies and overhang stabilities. To explain those observations, we present here experiments involving one or two substrates (overhangs) only. When performing GGA at a stable temperature of 37°C, we found that GGA efficiency strongly correlates with overhang stability. Combining those experimental results with a kinetic model, we were able to determine how relevant parameters (time, temperature, molarity, stoichiometry, stacking energy) influence GGA. This work provides a comprehensive view of low-assembly GGA, defines the required optimal conditions and substrates (allowing the fabrication of constructs for single-molecule experiments with unprecedented yields) and gives new insights into DNA ligation that are crucial in biology.
Functional materials are challenging to characterize because of the presence of small structures and inhomogeneous materials. If interference microscopy was initially developed for use for the optical profilometry of homogeneous, static surfaces, it has since been considerably improved in its capacity to measure a greater variety of samples and parameters. This review presents our own contributions to extending the usefulness of interference microscopy. For example, 4D microscopy allows real-time topographic measurement of moving or changing surfaces. High-resolution tomography can be used to characterize transparent layers; local spectroscopy allows the measurement of local optical properties; and glass microspheres improve the lateral resolution of measurements. Environmental chambers have been particularly useful in three specific applications. The first one controls the pressure, temperature, and humidity for measuring the mechanical properties of ultrathin polymer films; the second controls automatically the deposition of microdroplets for measuring the drying properties of polymers; and the third one employs an immersion system for studying changes in colloidal layers immersed in water in the presence of pollutants. The results of each system and technique demonstrate that interference microscopy can be used for more fully characterizing the small structures and inhomogeneous materials typically found in functional materials.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.