The purpose of these series of experiment was successful as seen from the results. The recombinant form of the green fluorescent protein was successfully expressed and purified from E. coli. Though a high yield and purity were not obtained, the qualitative monitoring of the rGFP activity was consistent with the quantitative values obtained. Qualitatively, the elution fraction E2 fluoresced the most when placed under a hand-held UV lamp. Quantitatively, E2 also had the highest activity with 41900 RFUs when read by a fluorescent microplate reader. One way to increase the yield of rGFP would be to increase the IPTG induction time. This would increase the amount of rGFP that is expressed and consequently increase the amount of rGFP that is purified. There were other things during the experiment such as contaminant proteins that could also have affected the yield of rGFP obtained. …show more content…
GFP or rGFP has several unique characteristics which makes it a good research tool. rGFP is a small protein that needs no cofactor for fluorescence and detection of this protein is also easy and inexpensive. As a follow-on experiment, how GFP can be used as a tag for proteins can be explored. In this experiment, we expressed GFP in E. coli. As a follow-on experiment, we could express GFP in other organisms and use that as a tag to investigate gene expression. GFP would be expressed in three marine bacteria from a lac or npt-2 promoter (Stretton, Serina et al.). The bacterial strains would then be tagged with GFP which enables the visualization and the monitoring of gene expression in living cells (Stretton, Serina et
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In the second part of the experiment, they took 3 test tubes, one containing a small amount of potassium chloride, one with a like amount of potassium chlorate, and one that contained a solution obtained from adding distilled water to the crucible, that was used in the first part of the experiment, and heated it for about a minute, and then they added 10 ml of distilled water to each and stirred. In each of the tubes, they added 5 drops of dilute (6 M) nitric acid and 5 drops of 0.1 M silver nitrate solution, then stirred each test tube and observed carefully.
The mole is a convenient unit for analyzing chemical reactions. Avogadro’s number is equal to the mole. The mass of a mole of any compound or element is the mass in grams that corresponds to the molecular formula, also known as the atomic mass. In this experiment, you will observe the reaction of iron nails with a solution of copper (II) chloride and determine the number of moles involved in the reaction. You will determine the number of moles of copper produced in the reaction of iron and copper (II) chloride, determine the number of moles of iron used up in the reaction of iron and copper (II) chloride, determine the ratio of moles of iron to moles of copper, and determine the number of atoms and formula units involved in
Green fluorescent protein (GFP) comes from the jellyfish Aequorea Victoria is rare proteins with high fluoresce and absorbance. The purpose this experiments is to purify and express a His2-tagged recombinant from of GFP (rGFP) from the E. coli strain BL21(DE3)< pRSETA-GFPUV > through a series of experiments by using Ni+2 agarose affinity chromatography technology. The GFPuv gene (UV-optimized GFP) was over expressed in the E. Coil strain BL21 (DE3) (pLysS) as an n-terminal His6/Xpress epitope tagged bind protein. Then using Ni2+ Agarose affinity chromatography to obtain purification of the crude extract. Then observe under the long wavelength UV light, the activity of the rGFP in the column fraction. Bradford assay was performed to obtain the total protein amount. When calculating the
If we are creating codons that are creating more of the specific amino acid this ultimately leads to there being a slightly higher emission peak. The most common form of modified GFP is known as S65T because it has a higher emission yield which will cause more fluorescence. With that being said yeast enhanced GFP, or yEGFP, is a different form of modified GFP which can reach an even higher emission peak then S65T. In order to amplify yEFGP, the needed sequence went through PCR where the restriction eznymes Pac1 and ASC1 were place on both sides of the PCR product allowing for the PCR product and pWDH444 to have the same sticky end sequence. The same enzymes were used two cut the S65T gene in order to move in yEGFP.pWDH444 is a plasmid that contained the RAD54 promoter. Since GFP was connected to the promoter polymerase could attach and go downstream and effect GFP. The area between the restriction enzymes Pac1 and Asc1 is where S65T is being cut and replaced with yEGFP. Another feature of pwDH444 is KanMX3, which provides resistance to kanamycin/G418 as well HO, which is the yeasts gene fragment. 2 microns are also present in order to aid with the replication process. The major advantage to the new yEGFP plasmid is that it increases the light output by more than double of S65T, which simply, allows for better GFP detection. The new plasmid allowed for the GFP signal to be more easily and identified by a fluorescent scans and its sensitivity improved many ways. The reason it could not previously allow whole cell measurements and was causing weaker sensitivity was due to a weaker output signal and too much light scattering. A more sensitive flourimeter was added as well as a sharp cut-off filter between the chamber and light emission detector allowing for both less light scattering and a strong reader of output. Multiple
Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).
Chemistry is composed of many molecules and changes of properties in the matter of science. The elements on the periodic table arranged from metal to non metals, plays an important role when it comes to making a compound and mixture. Each of the element have their own unique atomic mass which matters when it comes to organizing the periodic table and when there is a calculation. There are lots of importance about the atomic mass of each element that will be discussed later in this essay. An experiment performed using the mass of zinc and copper has determined whether which element have excess remaining. Will there be a difference if an alkali metal was used?
The petroleum lab and environmental reflect the major focus of the lab well. The abstract concisely communicates the purpose of the lab, the approach, the results, and the significances of the findings. The electrical lab doesn’t have an abstract but sometimes instructors do not require an abstract section. The environmental lab does a better job at writing the abstract because it’s shorter and more direct to the point, therefore easier to understand.
Furthermore, the PCR product will then be purified, in order to digest and ligate into a vector and transform E.Coli cells, in later parts of the experiment, to induce bioluminescence in those E.Coli cells .
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.
GFP was first isolated in in the 1970’s from the jellyfish Aequorea victoria. Scientist were able to use the genetic engineering to introduce fluorescent proteins into living organisms once identifying its specific DNA sequence. Osamu Shimomura, Martin Chalfie and Roger Tsien discovered the different changes in the patterns of absorption and emission in order to develop a rainbow fluorescent protein. At this time, the scientists notices that amino acid substitutions in GFP changed the behavior of the protein responsible for light production or a chromophore. The GFP and its related fluorescent proteins have become an essential tool in cell and molecular biology. proteins can be tagged with fluorescent proteins and then expressed in cells using DNA cloning strategies. Scientist regulate the expression of recombinant proteins using an inducible promoter or a genetic on / off switch. these sequences allow precise control because expression of the gym only turn on in the presence of a small molecule. In this experiment, and inducible promoter is used to regulate GFP
A positive control is when you test your experiment against something where you know what the effects will be. a negative control would be when you test the experiment with something you know will have no effect.
A wide range of biomolecules such as enzymes, microorganisms and receptors as well as either whole or attenuated animal or plant cells have been utilised as biological sensing elements. Microorganisms offer the advantage of ability to detect a wide range of chemical substances, can be easily genetically modified, and possess a broad operational pH and temperature range, making them ideal as biological sensing materials. (Nivens et al., 2004). Bioluminescent bacterial biosensors are of particular interest, bioluminescent bioreporters have become popular, because the bioluminescent response is easily detected and the assay need not be destructive to the cells. Thus, the bioreporter can be continuously monitored in real time. (Simpson et al., 1998).
Complicated processes of synthesis, folding and activation cause difficulties to achieve active TGF-β1 from expression systems. Most previous studies were performed to express the full length of TGF- β1 in mammalian cells and baculovirus-insect expression systems [11, 21, 22]. However, as the major secreted proteins by theses cells, consisted of non-covalently associated propeptide with the mature form of TGF-β1, the yields of biologically active purified proteins were not sufficient, and significantly decreased after removal of LAP propeptide . Therefore, some attempts were made to express biologically active TGF- β1, using the DNA sequence, encoding the mature domain in eukaryotic and prokaryotic systems. However, the expressed proteins were not active, and in some cases, chemical refolding was required, to maintain the biological activity [9, 10]. Here, we showed that the expression of biologically active TGF-β1, without the requirement of LAP coding sequence and chemical refolding, is feasible in yeast expression system. In addition, one limitation for the accumulation of mature TGF-β1 in the reported expression systems, was the low stability of the expressed protein, resulted from the activity of endoproteases. To overcome this problem, we used the engineered PichiaPink, strain 3, knocked out for protease B. Similar to higher eukaryotes, secreted proteins in yeast can be processed