"
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
Proteins responsive to light called photoreceptors are present in plant cells and its main function is to provide information about the environment, about circadian, seasonal and positional information. This information is crucial in plant development, given that it controls many processes of the organism such as germination, seedling development, sleep movements and many others (Mathew, 2006 http://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.2006.03051.x/pdf), and also, activate or regulate some cellular pathways, like chloroplast movement, cytoplasmic motility or endoreduplication
Phytochromes are some kind of photoreceptors sensitive to different shades of red light, with a range of reception between 620 and 740 approximately, including deep red and far red light (∼660 nm and ∼740 nm wavelength). Because of its nature, and the difference with other plant pigments, that only absorb wavelengths lower than 700 nm, these molecules are specific for detecting small changes in environment, like detecting the the length of the day or the relative position to other plants. (Mathew, 2006)
PIF6 construct (PIF6-TetR BD-NLS) and Phytochrome-B construct (PhyB-VP16 AD-AGS-NLS-PolyA) are both attached to the same promoter, so the expression of both constructs is equal and the concentration of both proteins is the same. In the cytoplasm, these proteins are found floating free in the cytosol when the environmental conditions are normal.
However, absorption of photons of deep red light (660 nm) makes the proteins form a complex, thus, going through its active form, and switching the system to its ON state. Alike, if cells are exposed to far red light (740 nm wavelength) the system could be permanently turning to the off state (Mueller et al, 2013)
Phytochrome-PIF6 complex in its active form triggers the transcription of two small interference RNAs which are complementaries to two RBSs presents in the de-greening construct, thus, shouting off permanently this system.
