GFP Practical Report
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Published: Tue, 12 Sep 2017
GFP is very useful as a reporter protein. After its discovery in 1962 its practical applications were put into use 30 years later by adding the coding DNA of GFP before the stop codon of other proteins. This allows for an easily detectable marker of the proteins presence without needing additional cofactors or causing any harm to the organism. The spectral characteristics of GFP can be changed by making mutations to the protein. In this investigation a Y66W mutation was made to wildtype GFP in order to produce a shorter excitation and emission wavelength. The mutation was made using QuikChange site directed mutagenesis. The protein was then cloned into BL21(DE3) pLysS for expression. The cells were then lysed and applied to a Ni-NTA column. This fractionated the lysate in order to analyse these fractions using SDS-PAGE, fluorescence and Bradford assays. It was found that the Y66W mutation was successfully added but due to another mutation in the stop codon additional amino acids were added to the C terminus of the protein. It was also found that purification was partially successful as GFP was eluted in the correct fraction. This is supported by the Bradford and fluorescence assays.
The green fluorescent protein (GFP) is a 238 amino acid protein with a molecular mass of 26,870 Da. It was first isolated from the jellyfish species Aequorea Victoria by Osamu Shimomura in 1962 (1). GFP is expressed in small photoorgans that are situated in the umbrella of the jellyfish. Douglas Prasher first realised the potential of GFP as a reporter protein (2). As proteins are smaller than the resolving power of electron microscopes, Prasher thought the GFP gene could be added into the gene for haemoglobin before the stop codon. This would allow the protein of interest to maintain all of its functions but would have the GFP protein at its C terminal end. This means that detection of GFP fluorescence would also indicate the presence of haemoglobin. Furthermore, GFP does not require additional cofactors or substrates to fluoresce. This means that it works extremely well as a non-invasive method of detection of protein expression. GFP is also non-toxic so it is able to be used in vivo without causing damage or harm to the organism.
Crystallisation studies (3) have shown that GFP has a barrel structure with the chromophore buried in the centre. This chromophore is comprised of 3 amino acids (Ser 65-Tyr 66-Gly 67) that undergo a series of spontaneous cyclisation reactions to create the active chromophore. Wild type GFP has a major excitation peak at 395 nm and a minor one at 475 nm with an emission peak at 509 nm. In vivo GFP is coupled to the protein aequorin which induces a blue glow when it interacts with Ca2+ ions and breaks down luciferin. This light is able to excite GFP and cause fluorescence. In vitro this is not the case, however GFP fluorescence can be easily induced by irradiating GFP with UV light.
As with all proteins, GFP can be mutated. By mutating key residues, such as residues in the chromophore, it is possible to change the characteristics of GFP’s fluorescence. The first of many mutations was the S65T mutation (4). This mutation improved the characteristics of the protein including increased photostability, fluorescence and a shift of the major excitation peak. This investigation is based on the engineering of GFP to create a mutant of GFP with a shorter excitation and emission wavelength by inducing the Y66W mutation. The aims of this investigation were as follows. To carry out site directed mutagenesis of GFPuv to clone into pET28c and transform the products into XL-1 super competent cells. Extraction of the plasmid after incubation overnight to check the purity and concentration of DNA. Preparation and transformation of BL21(DE3) cells. Lyse these cells and fractionate the lysate to purify his tagged GFP using a Ni-NTA column. Finally, detection of purified GFP by SDS-PAGE, Bradford assay and fluorescence.
The workflow of the investigation can be found in figure 1 in appendix 1. A more detailed protocol can be found in the BIOC2302 semester 2 practical manual on pages 6-15 with rationale for all experiments.
In site directed mutagenesis I 31 μl of water was added to the PCR reaction to give a total reaction volume of 50 μl.
In site directed mutagenesis II a supplied culture of cells was used in the experiment rather than cells from the transformation colonies in site directed mutagenesis I.
His tagged GFP was used instead of the mutant in the protein purification experiment in order for easier administration as the process is the same.
Site directed mutagenesis I
Before the wet lab work began it was first necessary to design primers for QuikChange to induce the Y66W mutation into the wild type GFP. These can be seen as figure 2 in appendix 2. These were created using the QuikChange primer design tool on the Agilent website.
The site directed mutagenesis was carried out using the primers supplied to induce the correct mutation. The products of this were cloned into the pET28c plasmid and the XL-1 super competent cells. The cells were plated as per the BIOC2302 practical manual and left to incubate overnight.
Site directed mutagenesis II
Upon checking the plates in the next session it was found that no transformed colonies had grown so a new culture was supplied. The undigested plasmid control grew approximately 50 colonies
The culture of BL21(DE3)pLysS cells was set up and the OD600 were recorded. They can be seen in table 1 in appendix 3. Within 50 minutes the culture had reached an OD600 of 0.483 meaning the cells were at the correct density for lysis.
The cells were prepared as per the BIOC2302 practical manual and the recombinant plasmid was extracted. The concentration measured was 121.7 ng/μl and the A260/A280 was 1.86 using nanodrop. Therefore, the ethanol precipitation was not carried out.
To prepare for sequencing 4.11 μl of this solution was diluted, with 5.98 μl EB buffer, to the correct concentration. This was then sent to be sequenced, the results of which can be seen in appendix 4. The primer has been highlighted in green and is surrounded by a box with the mutated codon in red. A deletion also occurred in the stop codon of the mutant as highlighted by the second box with deleted bases highlighted in blue.
The plates were inspected in the next session. It was found that the 200 μl transformation plate grew 3 colonies and the 50 μl transformation plate grew none. Transformation efficiency can be calculated for the 200 μl plate as 37 transformants/μg of DNA.
The cells were weighed and found to be 0.539 g so 2 ml BugbusterTM used. After lysis and fractionation the SDS-PAGE samples of each fraction were prepared and loaded onto the gel.
The Bradford assay was carried out while the gel ran. The BSA standards were calculated and the contents of each standard well can be seen in table 2 in appendix 3. The fractions were then diluted into their wells and the contents can be seen in table 3 in appendix 3. The plate was filled according to the map in figure 2 in appendix 5. The plate was ran and the absorbances for the BSA standards were taken from the plate readout and inputted into table 4 in appendix 5. From here a calibration graph was set up using GraphPad Prism and can be seen as graph 1 in appendix 6. This graph shows that the data points for the standards do not fall near the line of best fit.
The absorbance results from the plate readout for all of the fractions were imputed into table 5 in appendix 7. The equation of the line from graph 1 was then used to calculate the concentration of protein in each of the fractions. All of these values were also inputted into table 5.
With the Bradford assay complete the SDS-PAGE gel was disassembled, stained and a picture was taken. A map of the gel can be seen as figure 3 in appendix 7 and the picture of the gel can be seen as figure 4 in appendix 8. By looking at the picture it can be seen that in lanes 2, 3, 4 and 9 there are dark bands spanning the entire lane. In 5, 6 and 8 there is faint banding across the well. In well 7 there is a distinct small band in between the 25 kDa and 37 kDa molecular markers. Lane 8 shows no bands at all.
Finally the fluorescence assay was carried out as per the map of the microtiter plate in figure 5 in appendix 7. The results from the plate readout were inputted into table 5. From here a graph comparing the log of protein concentration compared to fluorescence of each fraction was plotted and can be found as graph 2 in appendix 6. This shows elution 1 with the highest fluorescence and the unbound x10 had the lowest. However, when comparing protein concentration the unbound fraction had the highest and wash 2 had none. Percentage fluorescence was also calculated and inputted into table 6 in appendix 9.
The first aim of this experiment was to transform the site directed mutagenesis products into XL-1 super competent cells. The correct primers were used in order to induce the Y66W mutation into the parental DNA. However, no colonies that were meant to take up the mutated plasmid grew but the undigested control grew around 50 colonies. This means the cells did not take up the plasmid because otherwise they would have grown on the plate. This could be due to a mistake made in making the PCR reaction mixture or the DNA may have become damaged at some point in the experiment. Additionally, the suppliers of the XL-1 super competent cells advice to avoid large changes in temperature. This was unavoidable in this experiment and may have contributed to the cells not taking up the plasmid. In the future more care should be taken while plating and preparing the cells. Also preparation of any reaction mixtures should be checked very closely in order to ensure the correct reactants are added in the correct amounts.
In site directed mutagenesis II the cell culture was lysed when the OD600 was 0.483. That is because E.coli cells are most likely to be made competent when they enter early log phase. This corresponds with an OD600 of 0.4-0.5.
The DNA concentration extracted in this experiment was found to be 121.7 ng/μl and an A260/A280 of 1.86. This means that the DNA is good quality as the desirable range for A260/A280 is 1.7-2.0 and the concentration was much higher that what was required. However, in future experiments to test for reliability multiple results should be taken. Furthermore, the data could have been confirmed by using the spectrophotometric method alongside using nanodrop.
The sequencing results in appendix 4 confirmed the successful incorporation of the Y66W mutation into GFP, creating the CFP mutant. However, the second mutation at the stop codon deleted 2 bases including the first base of the stop codon. This means that when the protein is expressed the ribosome will not stop and instead will continue to add amino acids onto the C terminus of the mutant until it reaches a new stop codon. There 144 bases between the original stop codon and the next in frame stop codon meaning 48 additional amino acids will be added to the C terminus. This codon can be seen highlighted in purple below the original stop codon. These additional amino acids could affect the folding or could increase the likelihood of aggregation of the mutant protein.
In the protein purification experiment the 200 μl transformation plate grew 3 colonies and the 50 μl transformation plate grew none. The transformation efficiency on the 200 μl plate was 37 transformants/μg of DNA. The reason why this is so low could be due to a number of factors such as the plating technique or the cells may not have been left to chill on ice for the optimum amount of time. However, the negative control did not grow any colonies, confirming that all of the bacteria on the transformation plate were transformed. Again, more steps should be taken if this was to be carried out again to ensure that proper plating and prepping protocol is followed.
The Bradford assay shows that in wash 2 there was no protein in the well. This means that any protein found in elutions 1 and 2 should all be His tagged GFP that bound the Ni-NTA column. This can be confirmed by the fluorescence results as the elution 1 fraction contained the majority of the total fluorescence with 44.13% of the total. However, all other fractions also produce some fluorescence. This could be due to GFP contamination in the other fractions. This could have occurred due to the resin being saturated, preventing further binding to the column. It could also be due to aggregation of the protein obscuring the His tag and preventing binding. Furthermore, the plots on the calibration graph do not fall on the line of best fit. This means that the equation of the line is not accurate and protein concentrations calculated using it are also inaccurate. Therefore, there could be more protein in each fraction than was calculated. This could account for the fluorescence in the wash and unbound fractions. The Bradford assay is quite limiting. This is due to the fact the assay only measures protein concentration rather than GFP concentration. This means that it is unsure whether the protein concentration measured in elution 1 and 2 is all GFP or it is contaminant protein. The same can be said for the other fractions, it’s unsure whether the protein concentration measured has been contaminated by GFP. In the future this assay should be carried out again to try and reduce contamination. The calibration graph should also be repeated until all of the data points fall on the line of best fit. Otherwise none of the calculated protein concentrations are accurate.
Finally, the SDS-PAGE results shows banding in wash 1, 2 and elution 1 and 2. This suggests that there is contaminating protein in all of these fractions. Elution 1 shows a clear band at approximately the 26-27 kDa mark as it is present just above the 25 kDa marker and is well below the 37 kDa marker. This suggest the band in elution 1 is GFP as it is the appropriate size and is in the expected fraction.
Another source of error could be due to the amount of pressure applied to the pipette. This will vary from person to person and will affect the volume of the solution being pipetted. As such small volumes were being used and there was a lot of solutions to be pipetted it is very possible a mistake was made. This mistake would have a big effect on the concentration and therefore could have a big effect on the absorbance values. These errors can be avoided in future by using the appropriate pipette for the volumes being used. Further reduction in errors can come from correct technique and by doing replicates and averaging values.
There could be some error in the microtiter itself. There may have been markings or scratches on the plate that weren’t seen at the time. This could affect how the light passed through the reader and therefore affect the absorbance values.
In conclusion, the aims of this investigation were to induce the Y66W mutation into wild type GFP using QuikChange site directed mutagenesis. Furthermore, the protein was to be expressed in competent BL21(DE3) pLysS cells. Finally wildtype GFP was to be purified using a Ni-NTA column and the fractions analysed with SDS-PAGE, fluorescence and Bradford assays. The investigation successfully introduced the Y66W mutation into wildtype GFP. However, the stop codon was also mutated adding an extra 48 amino acids on the C terminal of the protein. A band indicating the presence of GFP was found at the 26.9 kDa mark in elution 1, indicating it was bound to the column and was eluted. However, all factions were contaminated with other protein.
1. Shimomura, O., Johnson, F., and Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous. s.l. : J. Cell. Comp. Physiol. 59:223-39, 1962.
2. Prasher, D., Eckenrode, V., Ward, W., Prendergast, F., and Cormier, M. Primary structure of the Aequorea Victoria green-fluorescent protein. s.l. : Gene 111 (2)229-33, 1992.
3. Ormo, M., Cubitt, A., Kallio, K., Gross, L., Tsien, R., and Remington. S. Crystal structure of the Aequorea Victoria green fluorescent protein. s.l. : Science 273:1392-5, 1996.
4. Heim R, Cubitt AB, Tsien RY. Improved green fluroescence. s.l. : Nature. 373 (6516): 663-4., 1995.
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