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====Two Component System====
====Two Component System====
Revision as of 23:38, 4 March 2012
Molecules that respond to light are increasingly being used as input domains to facilitate the non-invasive control of variously complex gene circuits. The vast majority of light sensing proteins come from naturally occurring photoreceptors, although chemical photocaging has also found use. Since light can be coupled to a transmembrane receptor domain located on cell surfaces, it can be used in lieu of chemical signals to turn on or off signalling cascades.
Photocaging involves the covalent addition of a small molecule effector (e.g., IPTG or doxycyline) onto another molecule (the cage) that upon addition of light such as UV, releases the effector molecule to perform a task. The cage group typically consists of aromatic rings, such as ortho-nitrobenzyl moieties, that undergoes photolytic cleavage to unmask the target molecule. This permits the spatiotemporal control of gene function upon light stimulation. The caging of IPTG, for instance, permits the temporal control of genes under the control of the Lac operator. Alternatively, one might cage a small molecule within a protein blocking its activity, that upon light-stimulus permits the protein to function. Amino acids such as Tyrosine have been caged with the active site of a Polymerase, that upon light expression, permits gene expression. Alternatively, localization signals can be caged to augment controlled spatio control of proteins. 
Photoreceptors are naturally occurring multidomain proteins, found in all three kingdoms of life, used to relay signalling cascades from the cell surface to effector molecules. They are the largest class of proteins used. Photoreceptors contain a protein component and a photopigment, which reacts to light to undergo isomerization or reduction. This initiates a conformational change in the protein that is relayed to another protein on the inner cell surface, mediating a signalling cascade that can control gene expression.
To build a new cascade, one simply alters the protein-protein interactions between various photoreceptors coupled to new effectors. Effectors can transduce signal in various ways such as phosphorylation (kinases) or relocalization to the nucleus (transcription factors).
Types of Photoreceptors
- rhodopsins- opsin bound to retinal chromophore, visual light used in eye cells.
- phytochromes- bilin chromophore, red light detection used by plants for circadian rhythm.
- xanthopsins- trans-p-coumaric acid chromophor, blue light avoidance response used by Ec. halophila
- cryptochromes- cryptochrome chromophore, blue-green light used by animals for circadian clock to seed germination in plants
- phototropins- Flavin chromophore (FMN), blue light for signal transduction in algae and bacteria
- BLUF- Flavin chromophore (FAD), blue light signal transduction in proteo- and cyano-bacteria
Two Component System
Photoreceptors rely on coupling to another protein such as a kinase to signal effects. This is generally known as a two-component system. Two component systems are best known in signal transduction, where they are coupled not only to light, but chemicals, osmolarity, pH, mechanotransduction, and hormones. The kinase phosphorylates itself or another protein, after a conformational change due the membrane spanning domain (the receptor). Other proteins (called second messengers) then recognize the phosphorylation and relay this signal to gene expression.
In 2005, a joint collaboration between the Ellington lab at Texas and the Voigt lab at UCSF led to the invention of "coliroids", bacterial photographs. blah blah. The system consists of two components: 1) a red light sensor that generates a signal 2) a color generator that takes in the signal
In the 2005 paper , the team fused the phytochrome Cph1 to the histidine kinase domain of EnvZ-OmpR (originally an osmoregulator), which then relays a stop signal to a constitutive LacZ reporter producing a black compound. They also had to insert the phycocyanobilin biosynthesis pathway for generating the phytochrome. In this particular system, light addition causes autophosphorylation of the phytochrome/kinase fusion that shuts off signalling to the LacZ reporter. Therefore, cutouts that shine light onto a bacterial lawn at various places can generate images.
The 2019 Oxford English dictionary definition of Optogenetics was fancifully written as:
"the branch of biotechnology which combines genetic engineering with optics to observe and control the function of genetically targeted groups of cells with light, often in the intact animal"
This means scientists can switch on brains cells simply with light. Optogenetics is having the biggest impact in the field of neuroscience, where it is used to activate biochemical processes in neurons at millisecond timescales in live cells or animals. Genes such as halorrhodopsin (a photoreceptor), react to light and trigger an action potential in neurons, activating the cells. These light reactive opsins are essentially ion channels. Delivery of light specific neurons is done through a fiber-optic cable into a mouse's brain, if done on live animals.
The devices used for this technique are generally encoded directly into the DNA using genetic engineering techniques. The types of protein devices used can be categorized into actuators, which drive light commands into processes, and the sensors, which emit signals as an output for study (ie, GFP).
- chARGe, P2X2, TRPV1, TRPM8, channelrhodopsin-2, LiGluR
- SPARK, halorhodopsin
- FlaSh, SPARC, VSFP, Mermaid
- cameleon, camagaroo, pericam, G-CaMP
- synapto-pHluorin, sypHy
Last year, researchers used viral delivery (rAAV) of channelrhodopsin-2 into the eyes of blind mice to restore many vision functions. 
- Drepper T, Krauss U, Meyer zu Berstenhorst S, Pietruszka J, and Jaeger KE. . pmid:21336931.
Lights on and action! Controlling microbial gene expression by light.
- Riggsbee CW and Deiters A. . pmid:20667607.
Recent advances in the photochemical control of protein function
- van der Horst MA and Hellingwerf KJ. . pmid:14730990.
Photoreceptor proteins, "star actors of modern times": a review of the functional dynamics in the structure of representative members of six different photoreceptor families.
- Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, and Voigt CA. . pmid:16306980.
Engineering Escherichia Coli to see the light
- Miesenböck G. . pmid:19833960.
The optogenetic catechism.
- Doroudchi MM, Greenberg KP, Liu J, Silka KA, Boyden ES, Lockridge JA, Arman AC, Janani R, Boye SE, Boye SL, Gordon GM, Matteo BC, Sampath AP, Hauswirth WW, and Horsager A. . pmid:21505421.
Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness.