Engineering success
Continuing the direction of last year's project, we have created a construction that makes possible the overexpression of two specific genes, Arabidopsis UVR8 and PAP1, that are naturally present in plants and participate in the defense against UV radiation.
DESIGNING OUR CONSTRUCTION: OUR SELECTED GENES
Firstly, before we proceed to explain how we create the multigenic construction, why have we selected these two genes?
Arabidopsis PAP1 gene
PAP1 is a transcription factor naturally present in Arabidopsis thaliana plants. We aim to overexpress this gene to increase the phenylpropanoid and flavonoid pathways that stimulate the production of anthocyanins in plants. Anthocyanins are intracellular polar pigments whose main function is to protect plants against UV light due to their antioxidant property that avoids the production of free radicals.
This way, an increase in the levels of these photoprotective pigments could mean higher protection to UV-B light.
Arabidopsis UVR8 gene
The ultraviolet-B resistance gene expresses a seven-bladed β-propeller protein, which is a receptor of UV-B light. In the presence of white light, it exists as a dimer, its inactive conformation. However, the exposure to UV stimulates the dissociation of the protein into two monomers and generates a conformational change that enables the interaction of UVR8 with two proteins: COP1 and SPA.
The union of UVR8 with COP1 and SPA activates UVR8, which translocates from the cytoplasm to the nucleus, where it is functional, initiating a molecular pathway that leads to gene expression.
The formed gene expression is a photomorphogenic UV-B response which lastly results in the accumulation of UV-B absorbing flavonols.
Within this gene expression process, UVR8 monomers can re-dimerize thanks to the production and interaction with RUP1 and RUP2 proteins, repressors of UV-B photomorphogenesis, induced by the HY5 transcription factor. The UVR8-COP-SPA action is interrupted, adopting the initial conformation.
UVR8 Action
This way, the Arabidopsis UVR8 gene induces morphological responses that enable the plant to acclimatize and survive when absorbing radiation rays. So, the main objective when introducing this gene is to initiate another biological pathway in addition to the one started by PAP1, increasing, even more, the level of photoprotective pigments.
BUILDING OUR MULTIGENIC CONSTRUCTION WITH ARABIDOPSIS PAP1 AND UVR8 GENES
This construction was built in an easy way thanks to the GoldenBraid method that enables our module to be shared with the scientific community.
GOLDEN BRAID
Plant synthetic biology requires the adoption of a common standardized technology that makes the construction of increasingly complex multigene structures at DNA level easier, enabling the design of new organisms through genetic modification.
GoldenBraid is a tool for the assembly of genetic modules. Its simplified cloning schema with positional notation, or in other words, structured “grammar” facilitates the possibility of creating fully exchangeable genetic elements which can be shared between the scientific community, in any part of the world.
But, how are we able to do this? As the name suggests, the method consists of "braiding" the different constructions. Forming multigenic structures that will be eventually transferred into the plant genome. The designed constructions are inserted into two different plasmids. When the initial plasmid is an alpha 1 (α1) level, it will necessarily need to be combined with an alpha 2 (α2), subsequently resulting in the formation of an omega level plasmid, and vice versa, creating a continuous process.
The 4 pDGBs give a double-loop structure (braid) / https://gbcloning.upv.es/.
The GB method has been developed by Scientists from The Institute for Plant Molecular and Cellular Biology (IBMCP), a research center funded by both the Polytechnic University of Valencia (UPV) and the Spanish Research Council (CSIC).
Thanks to this method we were able to build up our construction containing Arabidopsis PAP1 and UVR8 genes. This way, GoldenGraid enables our module to be shared between the scientific community for future projects.
OUR CONSTRUCTION
Firstly, to create the biobrick containing UVR8, we used a pDGB1α1 vector in which we introduced the constitutive 35S promoter, the Arabidopsis UVR8 gene, and the NOS terminator. For this construction, we used an α vector to be able to join it afterward with the construction containing the Arabidopsis PAP1 gene created by the last team which is a pDGB1α2. As a result, as both of them were at an α level using the GoldenBraid system, we obtained a pDGB1Ω2.
Finally, in the last biobrick, we introduced the kanamycin resistance gene which is one of the most commonly used markers for the selection of transformed Arabidopsis. This enabled us to select those seeds which were transformed with our final construction, as we grew these transformed seeds on plates with this antibiotic. This way, only the seeds which had our construction would be able to grow on these plates. Joining the pDGB1Ω2 vector, containing our 2 genes Arabidopsis PAP1 and UVR8 genes, with a pDGB1Ω1 in which introduced the kanamycin antibiotic, we obtained a pDGB3α1.
Stages involved in the modular construction of pDGB3 α1 UVR8-PAP1-Kn.