GFP Protein Lab Report

The results of this experiment ended up being very conclusive and not positively or negatively surprising. The data and results collected we what we had expected more importantly the ones on the X-Gal plates where the colonies were above 100. Both the stains of E. Coli grew colonies when they were in the LB. When the ampicillin was present the cells did not grow even with the X-gal. The transformed cells on the other hand grew when the ampicillin was there. When the X-gal and AMP were present the transformed cells grew and had a blue tint, which is why we call it the pBlu lab. (duh)

In the LB (pGLO negative), it is expected to not see any colonies growing. As a result of this experiment, it shown any growth colonies but this only had shown a large number of white, circular colonies that were found across the surface of the agar. In the LB/Amp (pGLO negative), it is expected to see any growth colonies. In the experiment, the resulted was no growth colonies because this has Ampicillin and no pGLO. Now, the LB/Amp (pGLO positive), it is expected to have growth colonies in the agar plate. As a result, it was shown growth colonies in the agar plate because has positive pGLO regardless having Ampicillin. In the other hand, both positive pGLO have the same components but in one plate was added Arabinose. The LB/Amp/Ara (pGLO

Based on the samples the expression of rGFP was the greatest in the E3 both quantitatively and qualitatively. The purity of rGFP was however low. The rGFP protein was purified using the Ni+2 agarose chromatography. This part of the experiment did not achieve a high purity. Causes of this may have incorrect pipetting mechanism or incorrect amount deposition. Improving pipetting skills might have lead to more accurate results. A better comprehension of the purpose of each step in the protocol may have resulted in improved results. Also personally having to use GCE of a partner’s could have altered the results. Being more strict with the lab procedures, and not messing up with the timings of certain steps by accident would have improved data. Although minor errors may have occurred the greatest fluorescence occurred in wash fraction W4 and elution fraction E3 quantitatively, and this data matched the qualitative observation of the fluorescence in both these samples. W4 had the greatest protein amount. W1 was the void volume and only contained breaking buffer without rGFP therefore there should have been no fluorescence, and this occurred correctly. Samples W2 and W3 had slight fluorescence which could be explained by the rGFP lacking the His6 tag required to bind to the Ni+2 agarose, or all the binding sites in the column could

When lysing the cells, add 20 - 50 μl chilled lysis buffer (RIPA buffer with protease inhibitors and PhosSTOP), and keep the samples on ice for 30 min. Centrifuge the samples at 20,000 × g for 20 min at 4℃. Transfer the supernatant to new tubes,

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

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

In the pGLO Bacterial Transformation lab, Escherichia coli is transformed with a gene encoding green fluorescent protein by inserting a plasmid containing the GFP gene, beta-lactamase, and arabinose into the bacterium. Successfully transformed bacteria will grow in the presence of ampicillin and glow a bright green color under ultraviolet light. The sugar arabinose is responsible for switching on the GFP gene in the transformed cells, without it, the gene will not be expressed.

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

In our hypothesis we stated that only the container containing all of the components +pGLO, LB broth, ampicillin, and arabinose would be the one that genetically transformed. In order for the bacteria to grow at a rapid pace all it needed was LB broth but when you added ampicillin, an antibiotic, it killed off all of the bacteria. +pGLO has the gene to resist the antibiotic so when that was added it was allowed to grow but there was no sugar to turn on the glowing protein. Finally, after arabinose, a sugar, was added it turned on the switch located in the +pGLO for the fluorescence and enabled to grow and glow.

coli BL21(DE3)pLysS cells and a single colony in a transform agar plate was inoculated into LB broth containing 50µg/ml kanamycin. When absorbance reading at 600 nm reached approximately 0.5, the expression of His-tagged Δ gD1-Δ gD2 protein was induced by the addition of 0.5 mM isopropyl-1-thio-β-D-galactoside (IPTG) at 20˚C overnight. The recombinant protein was purified by affinity chromatography as described previously.(12) The concentration of purified recombinant protein was measured by BCA assay according to the manufacturer’s instruction. The level of endotoxin in each purified protein was quantified as previously

The reason why it did not inhabit any change after the experiment was conducted is because it had no plasmids added to support the growth and the ampicillin actually killed the bacteria. The plasmid would have served as the vector to insert the gene into the E. coli. The -pGLO LB plate thrived because there was no ampicillin present. While the +pGLO LB/amp/ara plate ended up glowing under the UV light because the GFP was only able to show with the presence of the arabinose, because it is what activates the operon. The +pglo LB/amp plate contained a few colonies but it did not glow under the UV light due to the absence of the

Placed it to the shaking incubator at 37˚C to prewarm. Heat shocked the cells for 60 seconds in 42°C water bath. Added 950 µl of warm (37°C) SOC medium and incubated with shaking (160-225rpm) for 50 minutes at 37˚C. Used two LB plates. Plate 100 µl of each transformation on a separate LB agar plate containing carbenicillin which is the antibiotic, IPTG to induce expression of lacZ and X-gal a substrate for β-galactosidase. The plastic L-spreader was used to distribute the bacteria.

If a gene that codes for Green Fluorescent Protein transforms bacteria and GFP glows when transformation occurs, then when two micro test tubes have 250 microliters of transformation solution and places in an ice bath, then 2-4 bacteria colonies are added to each tube with a sterile loop; then a plasmid (pGLO) is added to one of the tubes, incubated in ice for 10 minutes, then heat shocked for 50 seconds at 42 degrees Celsius, then back into 9ice for two minutes; then LB nutrient broth is added to both tubes (250 microliters) and set out at room temperature for 10 minutes. Then, 100 microliters of each solution in the tube are added to four

Genetic transformation occurs when an organism’s genetic makeup is altered due to the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab the pGLO plasmid is introduced into E. Coli bacteria, and incorporates the genes which code for the GFP and beta lactamase to the bacteria’s genome which as a result will be modified. To test the effects of the plasmid, bacteria treated with the plasmid were grown on separate plates, the first containing LB nutrient broth and ampicillin, another containing LB nutrient broth and arabinose and another containing LB nutrient broth, ampicillin and arabinose. Two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that did not contain the plasmid. Since the lab was about genetic transformation, the goal was to find which plate would glow. It was found that the plates that were not exposed to the plasmid did not glow, and the plates containing LB and arabinose and LB, ampicillin and arabinose did glow. The plates containing ampicillin, the antibiotic that kills E. coli did not grow whereas the remaining plates at least had some growth.

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.