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Team:BIOSINT Mexico/Switch
From 2014.igem.org
Reset - Red light switch
Description
We incorporate a red light-responsive gene expression system using far red (740 nm) light wave. This far red light sensor works as a switch that inactivates the expression of the de-greening system genes, is based on the interaction between the Phytochrome B and the phytochrome-interacting factor 6 (PIF6) from A. thaliana (Müller,K. et al, 2013).
In the literature, the switch used two different light waves, deep red (660 nm) for the activation, and far red (740 nm) for the immediate and permanent deactivation of the de-greening system. However, for this project, we designed a system responsive only for the far red light and which only function is the inactivation of the degreening of the plant.
The first three hundreds nucleotides of PIF6 (BBa_K1150005) are fused to the DNA-Binding Domain of the TetR (BBa_K909007) protein and attaches to its operator site upstream a minimal promoter (PCaMV 35S BBa_K788000), also, its fused with a Nuclear Localization Sequence (NLS: BBa_K1150010) and a polyadenylation tail (PolyA: BBa_K1150012). The activation domain was used in order to induce the system expression, as proposed by Müller,K. et al, attached to the DNA binding domain of TetR.
The first 650 amino acids (1950 bp) of the Phytochrome B (BBa_K1150004) protein are fused to an eucaryotic transactivation domain (VP16: BBa_K105001) from Herpes simplex virus, a 3 amino acid protein linker (AGS: Linker BBa_K1150013) and the NLS and PolyA, everything attached to the same minimal promoter (PCaMV).
In presence of far red light, the phytochrome and PIF6 complex will transform to its active form, and subsequently, it will activate the production of the small interference RNAs (RBS1 and IRES), that binds to the mRNA of the de-greening system at the RBSs positions, thus, stopping the binding of the ribosomes and the traduction of the messenger to protein. Also, on dark times this system can be turned OFF for several hours (Müller,K. et al, 2013)
The components of the PhyB transcription factor (PhyB-VP16-NLS and tetR-PIF6) were under the control of a minimal promoter, specifically, the Cauliflower mosaic virus 35S promoter (PCaMV 35S) to optimize the red light inducible gene expression. (Müller,K. et al, 2013) This promoter was selected because it is widely used on plant genetic engineering, because its strength and constitutive nature.
Light response
Phytochromes are photoreceptors that provide plants with circadian, seasonal, and positional information critical for the control of germination, seedling development, shade avoidance, reproduction, dormancy, and sleep movements. Phytochromes are unique among photoreceptors in their capacity to interconvert between a red-absorbing form (absorption maximum of ∼ 660 nm) and a far-red absorbing form (absorption maximum of ∼ 730 nm), which occur in a dynamic equilibrium within plant cells, corresponding to the proportions of red and far-red energy in ambient light. Because pigments in stems and leaves absorb wavelengths below about 700 nm, this provides plants with an elegant system for detecting their position relative to other plants, with which the plants compete for light. Certain aspects of phytochrome-mediated development outside of flowering plants are strikingly similar to those that have been characterized in Arabidopsis thaliana and other angiosperms. However, early diverging land plants have fewer distinct phytochrome gene lineages, suggesting that both diversification and subfunctionalization have been important in the evolution of the phytochrome gene family. There is evidence that subfunctionalization proceeded by the partitioning among paralogues of photosensory specificity, physiological response modes, and light-regulated gene expression and protein stability. Parallel events of duplication and functional divergence may have coincided with the evolution of canopy shade and the increasing complexity of the light environment. Within angiosperms, patterns of functional divergence are clade-specific and the roles of phytochromes in A. thaliana change across environments, attesting to the evolutionary flexibility and contemporaneous plasticity of phytochrome signalling in the control of development.
Photoreceptors function at the interface between organisms and their environments, providing information that is critical for the appropriate timing of growth and developmental transitions. The exquisite fine-tuning of land plants to their light environments is manifest in numerous phenomena, from the coordinated control by three distinct photoreceptor systems of branching in the filamentous protonemata of a moss (Uenaka et al . 2005) to the preconditioning by a single photoreceptor that enables germinating Arabidopsis seedlings to anticipate their most likely environment (Mazzella et al. 2005). Such responsiveness to environmental signals is useful only if it is not lost when new environments are encountered, and when plant form and life histories change. In order to promote survival, photoreceptor systems must be robustly linked to the signalling networks that ensure suitable responses.
At the same time, both information gathering and processing must be flexible enough to change when new challenges are presented. In the case of phytochromes, the principal photosensory function is to detect the relative proportions of red (R) and far-red (FR) energy in ambient light (Smith 1982). While this basic function has been conserved through millions of years of prokaryotic and eukaryotic evolution, the organisms in which they are found have diversified profoundly. The responses induced by light signals are concomitantly diverse, shaped by the morphologies, life histories, and environments of the photoreceptorbearing organisms.
The diversity of phytochrome-mediated regulatory functions in major clades of green plants (green algae and land plants) reveals how a single photosensory function has evolved to meet many specific needs and reveals the ecological importance of phytochromes for all plants. Phytochromes control cellular responses and tropisms such as chloroplast movement, cytoplasmic motility, endoreduplication, and nyctinastic movements, and of tropisms such as gravitropism, polarotropism, and phototropism (e.g. Wada & Kadota 1989; Kim et al. 1993; Haupt & Häder 1994; Hangarter 1997; Gendreau et al. 1998; Takagi et al. 2003).
However, it is the role of phytochromes in the major developmental pathways of germination, de-etiolation, shade avoidance, and flowering that is likely to have had the biggest impact on the establishment and ecological success of the major clades of land plants. A review of the literature reveals the surprisingly early appearance of several responses considered to be important in the ecology of angiosperms, including differential control of germination in open and shaded habitats, delay of development in the dark coupled with rapid developmental responses to light signals, and shade avoidance. Moreover, the gene phylogeny suggests that functional diversification in red- and far-red sensing, perhaps coinciding with increasing complexity in the light environment due to the origin of canopy shade, has been important in ferns, gymnosperms, and angiosperms.
