Follow-On Lab Report

The induction process for the activation of rGFP was vital to this experiment, to ultimately determine the presence and molecular weight of rGFP from crude extract. T7 RNA Polymerase activates the T7 Promoter, by binding to it upstream of the rGFP gene, in the presence of IPTG, allowing the expression of rGFP. The expression results from the transcription of the rGFP gene that produces rGFP mRNA, which is then translated to produce the rGFP. By inducing the IPTG activation of the DNA regulatory sequence T7 Promoter, as seen in Figure 1, the presence of rGFP became apparent under the UV lights after the Ni+2 Agarose Affinity Chromatography washes and elutions. The G3 sample, three hours post-induction of the IPTG sample, had the highest fluorescence at 14901 RFUs.

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

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

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).

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.

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

This indicates that a promoter responds negatively when there no inducer and positively when an inducer is present. A lot of glucose cause a cAMP-CAP complex which causes little expressions of the lacZ gene, therefore there is little ONP produced. For the X gal plate strain 1 and 2 and 3 shows that beta galactosidase cleaved to the X gal because of the deep blue color showing on the colonies. For the X gal + IPTG plate, only strain 1 and 2 have a blue

The plasmid first had to lose S65T-GFP so it could take up yEGFP. This was done by adding Pac1 and Asc1 to the plasmid to cut out the gene and leave the proper sticky ends for yEGFP to ligate to. A gel electrophoresis was then used to separate pWDH444 and S65T-GFP. The pWDH444 was cut from the gel electrophoresis and removed from the gel. After successful removal of the plasmid from the gel pWDH444 and yEGFP were ligated to create a new plasmid with the ideal GFP. The new plasmid, pWDH444-yEGFP was the ideal plasmid for yeast due to the enhanced GFP with a codon bias for yeast and it had double the light output of S65T-GFP. The difference in fluorescence was detected using a extra sensitive fluorimeter than normal and 495-nm glass, sharp cut-off filter. The filter reduced the amount of light shattering leading to an improved sensitive reading from the

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.

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.

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.

In this laboratory, the purpose of this lab was to allow the lab students to understand

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 [11]. 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

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).