Optogenetic Synthesis

Green Fluorescent Protein, produced by the bioluminescent jellyfish Aequorea victoria, is a protein that fluoresces green under ultraviolet light. Since its discovery, properties of the protein have been improved by mutations in the gene resulting in the expansion of its spectrum, which now contains brighter variants and multiple different colors. GFP is used in a wide variety of applications and technologies. Its many different applications have contributed greatly, and continue to do so, in numerous fields of study including, but not limited to, cellular and molecular biology, microbiology, biotechnology, and medicine.

The approach on minimal and maximal light intensity was thereby explored to determine if the Euglena cells are in fact light induced or thermally induced. The change in shape as hypothesized was light induced, and predominately reactive upon the maximum light intensity by an additional 10%. The theory of the Euglena cells being thermally induced was withdrawn, due to the fact that when the light source was minimized the Euglena cells reverted back to their “original” form. As stated in Evangelista et al. (2003), the cells are proven to be capable of regenerability. In further validation, the regenerability of the cells can stem from the pellicle which “provides an anchor to prevent displacement at both the anterior and posterior ends”, as stated in Suzaki and Williamson, (1985). Thereby confirming that the pellicles maintain their function under extreme photoresponse environments. However, this function can be impacted by the addition of 1% methyl cellulose. With the addition of 1% methyl cellulose the extracted cell models for both light exposures differed from that of earlier light induced pellicular movement. This decrease in the photoresponse rate in the active cells identifies that signal transduction pathway was impacted. As studied by Isekl, 2002, the photo activated adenylate cyclase, which can act as both a photoreceptor and effector catalyze cAMP synthesis, must be compromised in the step-up photohobic

On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded.

Scientists can study and manipulate genes by using precisely engineered plasmids, According to ADDGENE, a nonprofit plasmid repository, plasmids “have become possibly the most ubiquitous tools in the molecular biologist’s toolbox” (“What Is a Plasmid?”). In this experiment, we will use pGLO to genetically transform the bacteria E. coli. pGLO is a genetically engineered plasmid that carries the reporter genes for both green fluorescent proteins (GFP) and the ampicillin resistance. GFP, a protein typically found in the bioluminescent jellyfish Aequorea victoria, exhibits a bright green fluorescence in the presence of blue to UV wavelengths (Mecham); the protein absorbs UV light from the sun and emits it as a lower energy green light, exemplifying the second law of

The GFP gene is normally isolated from the jellyfish Aequorea victoria, allowing it to fluoresce with the absorption of ultraviolet light (Goodsell, 2003). This gene is inserted into a region of a plasmid to be regulated by an operon (Brown, 2011). In the plasmid, GFP is normally repressed but when arabinose is present, it will induce the gene to make enzymes able to digest the arabinose, and once there is no more arabinose,

Western blotting - In Western blotting first, the macromolecules have to be separated via gel electrophoresis. The molecules now separated by electrophoresis are blotted onto either a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane (a second matrix). To inhibit the binding of nonspecific antibodies to the membrane surface it is subsequently blocked. Then a complex is formed (a probe) from the protein that was transferred and an enzyme linked with an antibody. The enzyme is supplied a substrate then the 2 together should create a product e.g. chromogenic precipitate that can be detected. Detection methods with most sensitivity use chemiluminescent substrate because light is a by-product of the reaction between the substrate and the enzyme. The output of the light can be measured using a CCD camera or on the other hand, antibodies that have been tagged with fluorescents that are detected with a fluorescence imaging system can be used (Thermo-Fisher Scientific 2015).

Previously, the use of mass spectrometry has played an essential role in the identification of

Fluorescence microscopy based techniques are essential tools for measuring the dynamics of molecules in living cells and can be used to approach a variety of biological questions. The microscopy techniques applied in our lab can be subdivided into Time-resolved Fluorescence Anisotropy (TRFA) and Fluorescence Bleaching Techniques A combination of these complementary methods allows for a comprehensive analysis on multiple length and time scales. The microscope is intended to work in two modes, these are: single photon counting (two avalanche photodiode for measuring both parallel and perpendicular polarization of sample emissions) and imaging (ccd camera). The new design adds Fluorescent Recovery After Photobleaching (FRAP) to these current

Although essentially a broad term, the principal technology in optogenetics incorporates two key functions: light-responsive, control tools that can convey a function in the cell and customizable elements for delivering light to cells of interest; personalizing the control tools for use in the cell of interest; and acquiring analyzable data (Boyden, et. al., 2005). Optogenetically controlled systems had their beginnings in neuroscience, where the need to control a certain type of cell without altering others in the brain was the ultimate goal. This was the underlying concern with classic techniques used to obtain neuronal data; electrical stimulation could not be used for the targeting of individual cells, and the use of drugs to alter neuronal functioning is a slow, imprecise method that is potentially toxic to the cells. Fortunately, optical manipulation of neural activity was accomplished through a microbial opsin gene, a light-sensitive protein that allowed the neurons to react to specific wavelengths of light (Boyden, et. al., 2005). This “light-switching” was capable of being tested in freely motile organisms with no harm inflicted to the organism. Success with this particular group of proteins directed the research of optogenetics to the engineering of several variable

Rods are extremely sensitive to light and can react to a single photon. The light is absorbed by the pigment rhodopsin and the energy acquired results in decrease in cGMP which in turn causes the closing of Na+ channels. This hyperpolarizes the cell causing a release of synaptic vesicles.

‘Prostheses come in many forms; limbs, cochlear implants, dental implants and now the bionic eye is making its mark and a difference.’ (Dr. John D. Bissell, August, 2015)

In the field of neuroscience, the endeavor to develop technologies that allow minimally invasive, temporally and spatially precise, and genetically specific neural activation has occupied the minds of neuroscientists for decades. To this end, photostimulation techniques have been of particular interest. In the early 2000s, a set of light-gated proteins called channelrhodopsins (ChRs) from the green alga Chlamydomonas reinhardtii were characterized1,2 and noted for their potential ability to depolarize cells in other biological systems. In particular, channelrhodopsin 2 (ChR2), a light-gated nonselective-cation channel, has proven to be immensely useful. The genes expressing these photoreceptors have been used to introduce light-activated channels into selective cell populations of vertebrates to optically induce neural activity3.

“Light is a form of radiant energy that travels in waves made up of vibrating electric and magnetic fields. These waves have both a frequency and a length, the values of which distinguish light from other forms of energy on the electromagnetic spectrum.” (lightsearch, 2015). In a solar cooker it is not the suns heat that cooks the food, but the sun’s rays converted into heat energy. If the cooker is directly facing the sun and heat energy is efficiently retained by a lid covering, maximum effective cooking will be achieved.

Adamantidis 2015). It enables tunable manipulation of cell phenotypes using light (Jay & Barkin 2014). The technique employs a set of microbial ion channels and pumps that activate upon stimulation by particular wavelengths of light. After transgenic expression within neurons, these type I opsins permit optical modulation of membrane depolarisation or hyperpolarisation with temporal precision on the order of milliseconds (Snowball & Schorge 2015). The delivery of genes that encode proteins capable of conveying light sensitivity to neurons has provided a tool that may overcome some of the limitations of traditional neuromodulation techniques (Henderson et al. 2009). Srivats et al designed a method to optogenetically stimulate and inhibit acute pain in both normal and pathological states in freely moving nontransgenic mice by intrasciatic nerve injection of adeno-associated viruses encoding an excitatory opsin enabled light-inducible stimulation of acute pain, place aversion and optogenetically mediated reductions in withdrawal thresholds to mechanical and thermal stimuli upon transdermal delievery of optical stimuli. Techniques such as this, which eliminate the need for surgical implantation of stimulation devices, will obviously have a tremendous impact on the speed with which optogenetic therapy can

Halophiles have been commercially significant for centuries, with the earliest recorded case being that of ancient China and Middle East, where reddening of solar salterns as a result of growth of halophilic microorganisms was used as a biomarker of successful salt production (DasSarma et. al 2009). Today the role of halophilic organisms in salt production has evolved to facilitate rapid evaporation of salt crystals due to increased absorbance of light by their red-purple pigments. The purple membrane of the halophilic archaea that enables them to grow phototrophically and carry out phototactic responses contain the light-driven proton pump, bacteriorhodopsin, and sensory rhodopsins. Molecules like bacteriorhodopsin are especially of industrial interest because they are stable over a wide range of temperatures, usually between 0◦C and 45◦C, and pH values, usually between 1 and 11 (Ventosa and Nieto 1995). Moreover, the reactions of bacteriorhodopsin can be manipulated genetically, chemically or immunologically and are self-regenerative. Therefore, the ability of these biomolecules to convert the energy from light to chemical energy in a non-chlorophyll system has various industrial applications. For one, bacteriorhodopsin can be utilized as light sensors and for optical data processing or nonlinear optics. It is currently also being proposed for producing erasable photochromic film (Hampp et. al 1992). Furthermore, bacteriorhodopsin can also be used as a light sensor as they