Optogenetic Synthesis

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,

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.

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).

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

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.

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

Proteins tagged with fluorescent markers are extremely versatile tools in cell and molecular biology research. A fluorescent marker is, as the name suggests, a molecule that emits light of a particular wavelength following exposure to a photons of a shorter wavelength, for example a laser beam. If a fluorescent marker is a reasonable size and chemically stable enough to be attached to a protein, it is potentially useful to molecular biologists.

A major goal in neuroscience is to noninvasively, safely, and precisely be able to control specific neuronal populations. This

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

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

After voluminous amounts of studies, it is known that million years ago, the first human arose from our beloved ancestor, the Great Ape and just as humans arose, so has the way research can be approached. Molecular data can be used in modern phylogeny as a form to study evolutionary biology. This approach is practical because it uses extracted DNA and protein sequences to do an analysis on the sequence by finding similar sequences to it, in which help in phylogenetic reconstruction. In this study, two molecular markers were used: 16S rDNA and mtNCR (control region) to generate common ancestors among both markers. It was crucial to construct the phylogenetic tree to view the phenotypic evolution of the Great Apes.

‘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.

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.