Expanding GreenGate Cloning: A Modular Toolkit for Gene Expression and Functional Analysis across Multiple Heterologous Systems

Traditional molecular cloning techniques are time consuming, error-prone, lack high throughput, and usually only incorporate one DNA fragment into a vector at a time. To overcome these limitations, cost-effective alternative molecular techniques have been derived from the Golden Gate systems. GreenGate, one such system, allows the user to rapidly assembles six modules in a pre-defined order into a single plasmid, suitable for plant transformations. This study takes advantage of this concept, expanding it to broader range of heterologous systems, including single cells of Xenopus laevis and Agrobacterium tumefaciens-mediated transformations of Nicotiana tabacum, to facilitate on-going structural and functional analysis of membrane transporter proteins. Using standard molecular techniques, we successfully built several vectors, and validated them using transient transformation of N. tabacum with confocal microscopy to visualize the subcellular localization of fluorescent protein chimeras. A membrane bound signal of the compounded mCherry::Ma3L::HA tag and mCherry::Ma3H::HA tag was visualized at the periphery of N. tabacum leaf cells, which is consistent to previous lab findings regarding the protein localization of Ma3 within the tonoplast. For X. laevis expression vectors, one of the modules required to fully assemble the larger construct was consistently unable to be correctly synthesized. Current work is being continued to overcome this problem. Using this modular tool kit will help to streamline cloning protocols, resulting in greater experimental flexibility to be applied in a wider range of heterologous systems.

Participating in research in the Piñeros Lab through the BTI REU gave me hands-on experience in microbiology and expanded my range of research techniques. I gained confidence in troubleshooting experiments and making decisions throughout the research process, which helped me grow both personally and professionally. This experience helped me explore what I value in a future career- being able to talk to people about my work, share what I’m passionate about, and apply research to real-world problems. It also gave me the opportunity to network with graduate students and professors, which has helped me prepare for graduate school and find programs that align with my goals and interests.