This phenomena can be seen in aromatic biological proteins like tryptophan and tyrosine through the imidazole ring, allowing them to be synthesized in an array of environments Williams, Slatko and McCarrey, 2007). Fluorescence was first discovered in Aequorea victoria, which synthesizes aquorin, a chemiluminescent protein. It is a luciferase that catalyses coelenterazine oxidation through a calcium dependent reaction to emit blue light and green fluorescence under UV light. Soon after, it was cloned
Statement of Purpose Having pursued relevant projects, courses and considering my inherent ability, I find myself interested in life sciences and more specifically in Microbiology. I believe that pursuing Masters in Microbial Biotechnology at North Carolina State University will help me widen my knowledge, hone my technical and managerial skills and provide me an opportunity to be a part of the cutting edge research in the field and contribute to its growth in Industry. Consistently a top student
lab we introduced the pre-engineered pGLO into the E. Coli HB101 K-12 bacteria. This pGLO plasmid consists of the gene for the green fluorescent protein (GFP), the ampicillin resistance gene that inactivates the ampicillin in the LB media and the araC gene that indirectly controls the arabinose digestion enzymes [Fig 1]. Green Fluorescent Protein fluoresces bright green when exposed to UV light. The GFP gene only activates if there is arabinose present. When arabinose is not present, the arabinose
of the project was to study the non-fluorescent beta-barrel structured proteins and engineer its amino acid sequence to make them into fluorescent ones. Our approach was mainly based upon the studies of Green Fluorescent Protein (GFP) from a jellyfish Aequorea victoria. Tools like Python programming language, Gromacs, Ribosome, PROSS, and Pymol were used to study the structural components of non-fluorescent proteins. I gained invaluable knowledge about protein engineering and developed deeper fascination
Lab Report 2: PGLO TRANSFORMATION EXPERIMENT Doris Daniels PURPOSE: The purpose is to transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar. Also, it is to observe if bacteria will grow in the presence of ampicillin and without ampicillin using two –DNA plates and two +DNA plates. INTRODUCTION: In this lab experiment, we will perform genetic transformation by putting some new DNA into E. coli cells. When observing bacteria
efficiently passes longer wavelength (fluorescent) light. It is oriented at 45O angle to the incoming excitation light path and reflects the excitation light at a 90O angle directly through the objective and onto the specimen (Fig.1). It also reflects any scattered excitation light back in the direction of the illuminator. 4. Objective Lens: The excitation light is first focused on the specimen through the objective lens and then using that same objective the emitted fluorescent light is gathered and sent towards
transformation in naturally incompetent E. coli HB101. The pGLO plasmid was the means of transformation in this experiment. The pGLO plasmid contains three core genes: the bla gene encoding for ampicillin resistance, the gfp gene encoding for the green fluorescent protein, and the araC gene which activates the gfp gene in the presence of arabinose. The CaCl2 – heat shock method was used to transform E. coli HB101. After plating the bacteria, the petri dishes containing LB as a nutrient were incubated for 24
Figure 1 - Ponceau Stain blot Key : 1) Protein Molecular Weight Marker 2) Recombinant pET41(+) EGFP plasmid (positive control) 3) Non-recombinant pet41(+) EGFP plasmid (negative control) 4) Green colored clone properly expresses EGFP (green) 5) White clone that does not express EGFP (red) 6) Unknown sample (blue) 7) Purified GST-EGFP control A Ponceau stain can bind and identify all proteins. Lanes 2, 3, and 4 (our recombinant, nonrecombinant and green colony, respectively) have a slightly smeared
particle. When there is more than one, it is called a secondary particle. In order to measure these particles, they need to be suspended in a solution (Pruneanu, Coros & Pogacean, 2015). Dyed nanoparticles or internally fluorescent nanoparticles barely interact with cellular proteins which is what the study requires. They are also quite easy to manipulate. They can be easily internalized into cells and can be programmed to go to specific sites (Wolbeis,
can only magnify thin structures, therefore fluorescent microscopy are used to visualise the thicker structures. Fluorescent microscopy visualise the structures that emit light by allowing the light to get through the specimen. Methods Question 1: Briefly describe how immune-staining with an antibody is done. Immune-staining with an antibody is done by binding the antibody to a specific antigen. This is carried out by adding a staining fluorescent dye to a thin section of a tissue or cell.
In this experiment, the bacteria, E Coli, was transformed with the Green Fluorescent Protein (GFP). To start, the bacteria was grown, harvested, and added to a tube with -DNA. Calcium chloride was also added to the tube to allow the cells to take up the DNA and become transformed. Half of this mixture was then placed into a tube with +DNA, which contains the Green Fluorescent Protein, and both were placed in an ice bath for 10 minutes. When time was up, the tubes were placed in 42℃ water for 90 seconds
5th chromosome. By using a plasmid, the gene that encodes for DAT was attached to the gene for a Yellow Fluorescent Protein (YFP) in Human Embryonic Kidney (HEK) cells. This resulted in the production of the DAT and YFP proteins in the cells. Two antibodies were used to cause fluorescence in the cells. Background (Taryn) Dopamine Active Transporter (DAT) The DAT is a 12-membrane spanning protein that clears away excess dopamine from the synaptic cleft between neurons. Dopamine
in the E. coli cell, which allows the modified E. coli cell to begin to code for the GFP protein gene and the beta lactamase gene (ampicillin resistance gene). After modifying the bacteria cell, the changes involved with the plasmid were tested on 4 plates, two plates containing the pGLO plasmid (+) were treated with LB nutrient media. One of the LB plates contained arabinose in it, which should fluoresce green under UV light. The other LB plate contained ampicillin. Two other plates which did not
The green fluorescent protein (GFP) gene is a naturally occurring gene from a bioluminescent jellyfish. The gene allows for objects and animals to glow in the dark when activated by the presence of the sugar arabinose in the pGLO plasmid. The GFP gene is often used as a marker for gene expression and genetic transformation. The pGLO plasmid is a genetically engineered plasmid used as a vector in biotechnology to generate genetically modified organisms(GMO). M. Chalfie et. al. (1994) explain that
body structures during early embryonic development. Pitx1 in particular, encodes for a protein of some 283 amino acids (varying slightly in different species) that acts as a transcription factor and is in charge of regulating the expression of other genes involved in the differentiation and function of certain developmental regions. The scientists in this study have been interested in the role of this Pitx1 protein and the differentiation and function of the development in particular of the hind limb
(Sigma-Aldrich 2018). Transformation is used in many applications including the synthesis of essential proteins needed in the human body and to clone DNA needed to continue the process. (SLH 2007). In this experiment, E. coli will undergo the transformation process to insert a plasmid (pGLO) coded with ampicillin resistance, the ability to process arabinose, and the ability to synthesis green fluorescent protein (GFP). Ampicillin is an antibiotic that acts as an inhibitor on the E. coli’s ability
Introduction In every cell within an organism, the most crucial question is to survive or to die. In life, cell death is required so as to allow normal function. Cell death can be either physiological or programmed, in a process known as apoptosis. Cells that undergo apoptosis generally produce a wide range of morphological changes. These changes include shrinkage of cell, membrane blebbing, chromatin condensation and nuclear fragmentation. Apoptosis occurs due to the presence of a family known as
into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step. Introduction Enhanced green fluorescent protein (EGFP) was originally isolated from a bioluminescent jellyfish called Aequorea victoria. As suggested by the name, this protein fluoresces green when exposed to light in the ultraviolet range. The ultimate goal of the following experiment was to successfully create a pET41a(+)/EGFP recombinant plasmid that was
development are largely being recognized now, the theory behind optogenetics has existed for several years. In fact, opsins, proteins that undergo conformational changes when exposed to light, had been of interest to many researchers since the 1970s. In 2002, Gero Miesenböck’s lab modified neurons and showed that they could be stimulated by light. They employed a multi-protein system. Miesenböck is considered the founder of what is now known as optogenetics. Also in 2002, Ernst Bamberg, Georg Nagel
human gene. Now the plasmid, with the human gene, is reinserted into the bacteria. They are then cultured and produced in large quantities of identical bacteria carrying the human gene. Now, these bacteria produce the human protein coded for by the spliced human gene. The protein is then isolated and purified and are ready to be injected into patients (crop, etc.) (Gish 1998). Being able to do gene transfer gave us the ability to genetically engineer DNA and transfer it from one species to