Optogenetic Synthesis

PRACTICAL REPORT ON THE ISOLATION AND IDENTIFICATION OF CODEINE AND PARACETAMOLPRACTICAL REPORT ON THE ISOLATION AND IDENTIFICATION OF CODEINE AND PARACETAMOL AIM: To extract codeine and paracetamol from its tablet by solvent extraction and tentatively identify in comparison to standards using Thin Layer Chromatography. INTRODUCTION: Codeine or methyl morphine,

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,

Introduction: 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).

Introduction 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

Light is electromagnetic radiation, energy shown as a wave or a particle, that can be visually perceived as radiant energy.1 Light is measured by the wavelength and frequency. A wavelength is the distance from any point on one wave to the same point on the next wave and shown by lambda, λ.2 The different types of light begins with gamma rays and proceed to radio waves when increasing wavelengths are compared. Frequency is the number of crests per second and is expressed in hertz and shown as v. The pattern associated with light is indirectly related to the wavelength. The variability of light starting with gamma rays and ending with radio waves is connected by decreasing frequency. Frequency is independent of the wavelength and are both

channelrhodopsin enables deep transcranial optogenetic excitation. Nature neuroscience, 16 (10), 1499-1507. This paper focusing on a res-shifted variant of Channelrhodopsin shows that using red light can improve transcranial excitation because of the properties of red light. Red light is less scattered by tissue and is absorbed less by blood than previously used wavelengths. This development is very important for the potential use of optogenetics in chronic studies to treat neurological disorders, as cranial windows can be avoided. The opsin created is called ReaChR and has a spectral response to light at wavelengths greater than

Optogenetics is a technique where one can manipulate a gene in an organism through a light. This is made possible because of light sensitive proteins such as bacteriorhodopsin, halorhodopsin, and channelrhodopsin. Each of these proteins respond to light in different ways, such as pumping protons or chloride ions, allowing for different responses in the organism. Channelrhodopsin will be the protein used for this lab, where we determine, whether muscle contraction will occur in a Drosophila larvae that has been ingrained with the channelrhodopsin-2 channel and grown on ATR food, by using a program that will flash blue light at different intervals. ChR2 will act as an ion channel, allowing sodium to rush into a neuron and induce depolarization, create an action potential, and contract the muscle. Two plates, one containing larvae with ChR2 and one without the channel, was used as the subject where we then increased the MRR value in the program, allowing the blue light to flash at shorter intervals. This decreased the peristaltic waves produced from the larvae containing the ChR2 channel. Identifying unknown plates was also another focus in this experiment, as we are determining whether an unknown larvae plate has been ingrained with the ChR2 channel by observing its response to blue light. This is done by simply exposing the larvae to blue light.

Introduction: 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

5-week-old sdy mice and their WT littermates were injected with virus and implanted with optoelectrodes or optic fibers in the CA3 region, and then subjected to in vivo recording and the NOR test. (A) Experimental schedule. (B) NOR test protocol. (C) The site of viral injection and placement of optoelectrode. The image shows the expression of Cry2-Drp1 (red) and CIB1-Drp1 (green) and DAPI staining (blue) at the injection site. (D) Time-frequency spectrogram of LFP oscillations before and after object exploration during the test session of NOR. The dashed line indicates the time when the mouse started exploration. (E, F) Ratio of the gamma-range integrated power spectral density during novel object exploration to that during familiar object exploration (Gamma N/F) before and after light stimulation. (G, H) Ratio of the theta-range integrated power spectral density during novel object exploration to that during familiar object exploration (Theta N/F) before and after light stimulation. (I, J) Ratio of time exploring the novel object to that exploring the familiar object (Exploration N/F) before and after light stimulation. Data are presented as mean ± SEM; * p < 0.05, **p < 0.01.

ASAP1 was developed by inserting a circularly permuted green fluorescent protein into a voltage sensing extracellular domain. It’s believed that this design will have sufficient brightness, faster kinetics, as well as a dynamic range of detection of neuronal activity needed to make this ASAP1 design the ideal fluorescent based reporter for optical reporting of brain activity. This hypothesis was tested extensively in a laboratory setting to prove the claim.

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For this experiment we also made use of agarose gel electrophoresis, which takes a lot of time. Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory. In spite of the many physical arrangments for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated.