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The purpose of this experiment was to find out if a his6 tagged recombinant form of Green Fluorescent Protein (rGFP) could be expressed in a E. Coli bacterial culture. This was determined by purifying the sample through Ni 2+ Agarose Chromatography then finding the yield of total protein through a Bradford Assay. The purity of the GFP in the same sample was sought by analyzing the intensity of bands that appeared at about 31.5 kDa (the molecular weight of rGFP) in comparison to a ladder lane on an SDS-PAGE gel, while a contingent Western Blot allowed for a comparison of rGFP bands to a ladder to comparatively determine the molecular weight of rGFP. The Western Blot confirmed that the correct bands were analyzed in the SDS-PAGE gel for an approximate purity of 0.6, which indicated a yield of approximately 29.4 ug of rGFP for the third Elution fraction (E3) after a total protein amount of 49 ug was found by the Bradford Assay.
The GFP protein being used remains stable under changing conditions in temperature, pH, redox reactions, and the presence of chemical reagents, along with a half life greater than 24 hours. The formation of a chromophore at the center of this can shaped protein is what allows it to fluoresce, and requires no cofactors (2 pg. 352). At the center of the beta sheets that form the main cylindrical shape of GFP is the interaction of Ser64 and Gly67 on an alpha strand forming a five member ring which contains properties for fluorescence. When ultraviolet light strikes the chromophore region at a wavelength of 395 nm, a wavelength of 510 nm is reflected and seen as fluorescent green light. The excitation wavelength of 395 nm excites electrons and increases the energy of the protein. The protein loses energy as it reflects this emission wavelength of 510 nm is what is detected as given off by the protein. (2 pg. 351)
A restriction enzyme cuts into the pRSETA vector at sites that are complimentary to the translation start site and the Stop codon of the his6 tag sequence of rGFPuv. Prior to the translation start site, the t7 promoter works with t7 RNA polymerase to transcribe the his6 tag sequence of rGFP (2 pg. 359). This his6 tag sequence also contains an Expressed Antibody Epitope which allows for the binding of a primary antibody and then a second antibody which contains horseradish peroxidase (HRP) which is what makes the bands on the western blot luminescent (1 pg. 156-158). Histidine tagging is also what makes it possible for the GFP to be detected and purified away from other proteins that do not include rGFP due to its metal affinity for Ni2+ (1 pg. 95-96).
The 6 consecutive histidine residues have a metal affinity for the Ni 2+. This is why when the sample is passed through the Ni 2+ Agarose column, binding of the rGFP occurs with the column in the first wash steps. The elution steps occur with an elution buffer passed through the column once the his6 tag GFP has bound to the column. The elution buffer contains Imidazole which competes with the histidine residues for binding sites onto the Ni 2+ Agarose column (1 pg. 111-112). Elutions off of the column during the elution buffer passages, contain the purified rGFP. Purifying out this rGFP is essential to the purpose of this lab, because analysis of the his6 tagged proteins is needed to determine whether rGFP is expressed in the BL21<DE3>(pLysS) strain.
MATERIALS AND METHODS:
Bacterial Expression of rGFP
For the expression of bacteria, 2 bacterial cultures were used. One denoted as V, BL21<DE3>pLysS, pRSETA. The other denoted as G, BL21<DE3>pLysS, pRSETA-GFPuv. These were inoculated with LB (100ug/ml of ampicillin. 25ug/ml of cam). This was grown at 37 degrees Celsius with vigorous shaking until it reached an OD6000 equaling about 0.5. When OD600 has reached about 0.5 this indicates a time of zero, which is when 1 ml of each culture is to be pelleted into a 1.5 ml centrifuge tube. The supernatant is to be discarded, at which time these cultures are now labeled as V0 and G0 (time of zero), then stored at -20 degrees Celsius. The culture is then induced with IPTG at a 1mM concentration, and then continues to grow. The culture is then pelleted again into 1.5 ml centrifuge tubes (supernatant disposed) 3 hours after the induction of IPTG. These cultures are labeled as V3 and G3 (3 hours post induction) and then the pellets are stored at -20 degrees Celsius again. The G culture is then pelleted into 15 ml then labeled as G3-15ml and stored at -20 degrees Celsius. (1 pg. 105)
1 ml of breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) should be added to frozen bacterial pellet G3-15ml, then immediately pippeted up and down until the mixture is thawed and homogenous throughout. This should be similar to the consistency of mucous. After the sample has been thawed and is homogenous, it is transferred a centrifuge tube and should be vortexed for 5 minutes then placed in a 37 degree Celsius water bath for 10 minutes. The sample is then shaken in a dry air incubator at 37 degrees Celsius. Next the sample is centrifuged at 14000 xg at 4 degrees Celsius for 10 minutes. Finally the supernatant is obtained by pouring into another centrifuge tube, making sure to leave the unlysed pellet behind. (1 pg. 110)
Ni 2+ Agarose Affinity Chromatography
The Nickel Agarose column was prepared by first filling a 3 ml syringe with ¼ ml level of glass wool. A leur lock was filled with our breaking buffer [10mM Tris, pH 8.0; 150mM NaCl], then the syringe was filled with our breaking buffer and was allowed to drain. During the draining, the leur lock was positioned onto the syringed to prevent air bubbles. It was then opened to allow more drainage of the breaking buffer and removal of air bubbles. 1 ml of the Nickel Agarose slurry was deposited into the syringe column, then the leur lock was opened to drain the column until it was packed, then 5 ml of breaking buffer was applied to the column to wash out the ethanol in the slurry. The crude extract is then applied to the column and allowed to bind for 5 - 10 minutes. After this time, the column should be allowed to drain. The first two ½ ml elutions should be collected in 1.5 ml centrifuge tubes labeled as W1 and W2. 4 ml of breaking buffer should be added in ½ ml portions, which should be collected in labeled centrifuge tubes W3 - W10. After all washes have been collected, the column should be further washed with 5 ml of breaking buffer. Then 10 more elutions should be collected in labeled centrifuge tubes E1 - E10 by adding ½ ml portions of elution buffer [10mM Tris, pH 8.0; 150mM NaCl, 300mM Imidazole]. (1 pg. 111-112)
Total Protein Determination by Bradford Assay
In determining the amount of total protein, a reference was first constructed using known Bovine Serum Albumin in basic 1.05 ml Bradford reaction volumes. Known quantities of 0, 2.5, 5, 10, 15, and 20 ug were combined with the Bradford reagent, missed and incubated at room temperature for 10 minutes. These samples had absorbance read at 595 nm in the microplate reader, then a best fit line was plotted for the absorbance of the samples versus total amount of BSA protein. A Bradford Assay was performed in triplicate for wash fractions one through six and the elution fractions one through six. For those samples having an absorbance value lying outside of the standard curve, volume of sample was reduced from 50 ul to 10 ul of sample and 40 ul of water. Total protein was extrapolated using absorbance values for samples with the standard curve created from the BSA samples. (1 pg. 124-126)
rGFP Analysis by SDS-PAGE gel
A 12% resolving gel was prepared and poured and allowed to polymerize for approximately 30 minutes. A 5% stacking gel was poured on top of the resolving gel, along with an inserted comb to provide lanes in the stacking gel while it polymerized. Samples of G0,G3, GCE, W4, W3,E2, E3 were prepared by vortexing for one minute, boiling for two minutes, vortexing for one minute, and then centrifuging for a half minute. After the gel had been transferred to an electrophoresis tank, and the electrophoresis buffer was loaded, samples were loaded into lanes 1 through 7 respectively, and ran at 200 volts for approximately 45 minutes. Gels were then removed and stained with Coomassie blue. (1 pg. 111-112)
The Western Blot transferred the protein bands onto a nitrocellulose membrane by stacking a sponge and filter paper behind the gel and nitrocellulose which were facing one another. This was all compressed between locking cassette lids (2 pg. 375). Once the bands were transferred, the membrane was stained with Ponceau S for about 2 minutes. Once stained, the membrane was rinsed with distilled water letting the bands appear. Then the membrane was incubated in 5% non fat dry milk/TBS for a half hour. Then in triplicate the membrane was washed by incubation in 0.05% tween20/TBS and shaking for 5 minutes. Primary antibody, mouse IgG anti Xpress epitope Mab solution, was added to the membrane and incubated with shaking for 45 minutes. Another wash step was performed in triplicate exactly like the first one. The secondary antibody, sheep IgG anti-mouse IgG conjugated horse radish peroxidase polycolonal antiserum, was added and incubated with shaking for 45 minutes. Another wash step was performed in duplicate exactly like the previous ones, then for the third step TBS was used. The membrane was developed by adding TMB, then once bands were apparent the reaction was stopped by submerging in distilled water. (1 pg. 161-162)
The pRSETA-GFPuv vector arrives through repression of the lacI gene which represses the expression of the lac promoter. Bacterial cultures "V" (BL21<DE3>pLysS, Prseta) and "G" (BL21<DE3>pLysS, Prseta-GFPuv) are inoculated into 500 ml of LB to an OD600 of 0.1, and grown overnight at 37 degrees Celsius with vigorous shaking until OD600 had reached 0.5. 1 ml was pelleted from both V and G and stored at -20 degrees Celsius. These samples were induced with IPTG and then grown for 3 hours, when another pellet of 1 ml was taken from each and labeled V3 and G3, then stored at -20 degrees Celsius. After the bacterial chromosome was induced with IPTG the lacI gene was repressed and T7 polymerase was able to be produced. When T7 polymerase was produced more after induction of IPTG, more GFP was produced within the vector. Lysozome protein in the cell helped break down cell membranes along with the rupturing of cells during the expansion of freezing water, spilling out more strains containing GFP during the freeze thaw process.
Qualitative observations of the wash and elution fractions that ran off of the Ni 2+ Agarose column show that of the washes, the fraction with the most fluorescence is the sixth wash, while the third elution fraction retained the most fluorescence. The microplate reader indicated that the third elution fraction (E3) contained the highest fluorescence value of all the fractions. Using the absorbance values from the Bradford assays, total protein amounts were determined for the 3 microplate values by extrapolation from the earlier constructed best fit line using BSA protein. From those values an average value for the total protein in the E3 fraction was determined to be 49 ug.
In examining the SDS-PAGE gel a value for the molecular weight of the rGFP strand was first calculated to be 31.5 kDa. The band closest to this value in comparison to the ladder bands in lane 8 of the gel was designated as the band containing our strand. The ladder contained kDa values from top to bottom of 97.4, 66.2, 45, 31, 21.5, 14.4. This band in the E3 fraction containing rGFP, designated "B", was qualitatively estimated to have a purity of approximately 60%. Band B is 60% predominant in intensity over the other bands in the lane. The 60% purity of the total protein amount of 49 ug indicated an rGFP yield of 29.4 ug in the E3 fraction. The western blot shows that the band for rGFP appears at just below the 33.5 marker on the ladder. The ladder has kDa values of 108.5, 98.7, 54.6, 33.5, 29.5, 19.8 from top to bottom respectively.
Figure 1: Combined Elution Profile
Figure 1 is a hand drawn combined elution profile on the next page. The figure caption for the combined elution profile is belowâ€¦.
The column graph represents both the total amount of protein in the wash and elution fractions, and activity in terms of RFU's read at 595 nm. These fractions were obtained by eluting off of a Ni+2 Agarose Column first with a breaking buffer [10mM Tris, pH 8.0; 150mM NaCl], and were labeled W1 - W6. E1 - E6 elutions were obtained by use of an elution buffer [10mM Tris, pH 8.0; 150mM NaCl, 300mM Imidazole]. The elutions were obtained a week prior to the determination of total protein amount and activity by Bradford Assay. Error bars shown on the graph are very small in range and are difficult to interpret because they were drawn by hand.
Figure 2: SDS-PAGE gel
The SDS-PAGE gel above is a representative of purity of the rGFP strain in our fractions that have been run through gel electrophoresis. Lanes 1 through 8 contain fractions G0, G3, GCE, W4, W3,E2,E3, and a molecular weight ladder respectively. The molecular weight of the rGFP strain has been calculated to be 31.5 kDa, so it is estimated that in lane 7 band B is representative of rGFP because it is within the range of 31 kDa and 45 kDa in reference to the molecular weight ladder.
Figure 3: Western Blot
The Western Blot above represents the molecular weights of rGFP in the fractions G0, G3, GCE, W4, W3, E2, E3 in lanes 1 through 7 respectively in kDa. The molecular weight ladder in lane 8 shows bands weighing 108.5, 98.7, 54.6, 33.5, 29.5, and 19.8 kDa. Our rGFP bands are visible at a level slightly below 33.5 kDa across all of the fractions.
CONCLUSION AND DISCUSSION:
The results indicate that we were able to express rGFP in the E. coli bacterial strain. This is evident because of the Bradford Assay, SDS-PAGE gel, and Western Blot analysis of our Ni 2+ Agarose chromatography purification step. The Bradford Assay let us determine the total amount of protein in each of the fractions. From there the SDS-PAGE gel gave us insight as to just how much rGFP was in the fractions, more specifically the E3 fraction, by determining how pure bands were based on molecular weight. The Western Blot continued this type of analysis based on molecular weight, but it also used the binding of antibodies onto the Xpress Epitope on the rGFP strain.
In examining the SDS-PAGE gel, band B in lane 7 containing rGFP looked to be approximately 60% pure because of its intensity in relation to bands A and C. Band B appeared to be 60% predominant over the other bands in the lane, so from there the amount of rGFP in that fraction could be estimated by taking 60% of the total protein determined in that fraction from the Bradford Assay results. From 49 ug of protein in the E3 fraction, it was determined that 29.4 ug of that total was rGFP. This is also likely because of where band B lies on the lane in relation to the molecular weight ladder. Band B appears slightly heavier than 31 kDa but lighter than 45 kDa, which is a good range for it to fall since the molecular weight of rGFP was calculated to be 31.5 kDa.
The primary binding of mouse IgG, then the secondary binding of sheep IgG anti mouse IgG conjugated horse radish peroxidase allowed for a visualization of only bands containing rGFP in the Western blot. The primary antibody binds to the Xpress Epitope encoded in the rGFP strain, so we only see rGFP in the Western blot. This is also reassured with the bands appearances at just below the 33.5 kDa level on the molecular weight ladder. This suggests that it is close to our calculated molecular weight of 31.5 kDa.
The success of this experiment makes it now possible for GFP to be used as a tag for many purposes. Some uses might include the measuring of gene expression, the examination of intracellular movements, and the labeling and identifying of organism just to name a few. The ability of GFP to be used as a tag reveals possibilities that it may be used in other organisms besides bacteria. Follow up experiments to the successful expression of rGFP in a bacterial strain will be along the lines of labeling different unicellular organisms instead of bacteria. From there, the labeling and locating of different cellular components in other living organism could be attempted.