Description
Abstract
The development of inducible expression systems in plants is imperative to the field of synthetic biology. The University of Georgia’s 2018 iGEM team is expanding the iGEM registry profile of plant promoters and reporters. Here we report a modified Gal4/UAS system. The Gal4/UAS system is an inducible promoter system native to yeast that utilizes the Gal4 transcription factor to activate genes downstream of a minimal promoter enhanced by an upstream activator sequence (UAS). We have created a 6X UAS repeat combined with a minimal 35S promoter to provide enhanced expression of reporter genes such as GFP, AmilC, and the apoptotic initiator from bell peppers, BS3, in the model organism, Nicotiana Benthamiana. The introduction of these expression systems to the iGEM registry will enable future iGEM teams to produce targeted expression in plants with ease using a binary vector system.
Background
The GAL4/UAS system is a system for expressing transgenes in specific cells across a variety of organisms. The system is composed of two main parts: (1) GAL4 transcription activator (from Saccharomyces cerevisiae) and (2) UAS (upstream activation sequence), the enhancer sequence that the GAL4 activator binds to. In essence it is a binary expression system that is primarily used in Drosophila, mice and zebrafish. From a synthetic biology standpoint, one construct would contain a cell-specific promoter that drives the expression of the gene encoding for the GAL4 transcription factor. In a second construct, the gene of interest (transgene) would be regulated by the UAS. The two constructs will then work in conjunction to indirectly drive the expression of the transgene. The GAL4 activator will bind to the UAS promoter and subsequently facilitate the expression of the gene of interest. The indirect expression of the desired gene, as opposed to direct expression, can be attributed to one main reason: the UAS is known to express transgenes at much higher levels than endogenous promoters (Carter, Shieh, 2015). The BS3 resistance gene encodes a flavin monooxygenase that is activated by AvrBS3, and subsequently activates a plant avirulence response. BS3 and other proteins such as GFP and AmilC are being tested in our Gal4-UAS system with two different promoters to enhance the strength of iGEM's plant part registry.
Impact
Inducible Expression Systems are ubiquitous in synthetic biology, especially within the realm of the iGEM competition. As such, this year’s project strives to further strengthen a known expression system, which, in turn, will not only expand iGEM’s existing registry of plant promoters, but also allow future iGEM Team’s to easily express transgene using a binary vector system. Next year’s UGA iGEM Team plans to use this year’s project as a proof of concept to create a highly efficient, cost-effective biosensor.
