CLONING OF GFP FROM TRANSFORMED E.COLI CELLS
Asfaryani Rashid
18881828 Group B
In this study, expression of green fluorescence protein (GFP) on Escherichia coli was achieved via ligation using T4 DNA ligase, ligation buffer, BamHI and HindIII enzyme, vector pQE30. This gene encoding process transformed to pQE30-GFP and was screened to check for successful transformation. Plasmid mini-prep and restriction digestion were performed to confirm for transformation of E.coli cells then induction of the synthesis of hexahistidine-tagged GFP in cells transformed with pQE30-GFP. Immobilised metal affinity chromatography (IMAC) for purification of hexahistidine-tagged GFP. SDS PAGE was used to confirm the purity of GFP. Bradford Assay was done to determine the purity of GFP. Western blot analysis to further confirmed the identity of GFP (35 kDa). GFP could be used in a variety of applications such as developing live vaccines, analysing of polypeptide libraries, development of live vaccines, creation of whole cell allosteric biosensors, and signal transduction studies.(4)
Introduction
Green fluorescent Protein is a great tool in molecular and medical biology for its fluorescent properties which were used in localisation and expression (5). In this experiment, PCR was used to amplify ORF of the GFP which was derived from pe-GFP vector. Vent Polymerase, a thermostable DNA polymerase is used to ensure PCR results would not have mutations as it contained proofreading properties. Ligation between the purified and digested DNA and the digested vector that contained a bacteria promoter and a tag for hexahistidine-tag. After this ligation, the Ecoli cells were changed using PQE30-GFP construct, the new bacteria would be highlighted by the UV light. Its identity was then confirmed by the digestion with restriction endoclease. These cells were then synthesized by GPF and then purified by IMAC. This was brought to NCF and analysed by a blot of antibodies allowing the yield and purity of the GFP to be confirmed. This experiment hereby helped us understand the process to purify proteins.
EXPERIMENTAL PROCEDURES
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Amplification and purification of GFP coding sequence from pE-GFP
GFP sequence was amplified with Vent DNA polymerase (NEB, USA). Polymerase chain reaction was performed at a final concentration reagent of 10x Vent buffer (New England, USA), pE-GFP template (10ng/μl) with Primer E1; Forward Primer and Primer E2; Reverse Primer 5pmol/μl (integrated DNA Technologies, SG), 2.5 mM dNTPS (Promega, Madison, USA) , Vent polymerase ( New England , USA) into PCR tube.
Polymerase Chain Reaction was conducted and 3μl of PCR product was collected then, 2μl of 6 x DNA sample loading buffer and 5μl of DL water were added together with the sample. The sample was placed in the agarose gel so that electrophoresis (2) would occur. Remaining PCR product was purified using PCR purification clean up kit (Qiagen, california,USA) and eluted with nuclease-free water.
Ligation of GFP DNA and pQE30 Vector DNA
18μl of GFP DNA was transferred in a tube together with pQE30 and 2μl of 10x buffer were added to the tube, 5μl BamHI/HindIII premix (New England, USA) for digestion to occur at 37℃ for 2hrs. 5μl of 6x DNA sample loading buffer was added into each tube then, electrophoresis was performed on nu-sieve gel for digested GFP DNA insert and cut pQE30 vector DNA. 80μl of sterile water was added to GFP DNA insert and cut pQE30 vector DNA. DNA insert into the pQE30 vector was done by adding water, cut pQE30 vector, GFP DNA insert , 10x ligation buffer and T4 DNA ligase were added to 3 different ligases reaction which were vector plus insert , vector and insert only.
Transformation
0.5ml of Luria broth added to 5 tubes with 100μl aliquot of competent XL-1 blue E.coli cells with 2μl of pQE-GFP test (+GFP), vector only control (-GFP), insert only control,1ng/μl uncut pQE-GFP and water respectively . 200 μl of sample was transferred and spread onto agar plates for overnight incubation. +GFP and -GFP in a tube of 350ml of LB media were inoculated.
Induction of the synthesis of hexahistidine-tagged GFP in transformed cells
+GFP and -GFP 1.5ml was collected for mini prep.
50μl of sterile LB media added with 25mg/ml ampicillin stock for final concentration of 100μl/ml and +GFP cell suspension after 1hr of shaking record its OD reading on UV spectrometer (Shimadzu). 1ml of sample was collected and labelled as “0” after centrifugation.100mM IPTG to give a final concentration of 1mM were added. OD reading was recorded after 1 hour. 1ml of sample was collected and labelled as “+1” after centrifugation. After another hour, 1ml x 2 of sample into microfuge were collected after centrifugation and labelled as +2 (2hr post induction). The remainder of induced cells were transferred to a flask to 50ml screw capped plastic tube and centrifugation would occur at 4000rpm
NICKEL AGAROSE PURIFICATION OF HEXAHISTIDINE-TAGGED GFP
E.Coli cells were collected after 2 hours- post induction, were pelleted for 5mins at 1500rpm before it was resuspended using a lysis buffer(Disodium phosphate and sodium chloride at a pH of 8 with a lysosome 0.2mg/mL) and incubated at 37°C for 20mins. It was subsequently mixed for every 5 mins. The lysate was incubated with 3mM of MgCl2 and Dnase, with a final concentration of 1mg/mL were mixed occasionally at room temperature. Lysate was placed on iced and sonicated for 1 hour in a water bath. Crude cell homogenate was centrifuged and the supernatant was collected as crude cell extract. A sample is set a side for SDS-Page and and GFP quantification assay. 50% slurry of Ni-agarose beads was added to 1ml of crude cell extract in lysis buffer. It was mixed for 1 hour at room temperature and the supernatant was collected as “unbound protein”. The pellet was resuspended using the wash buffer (and the supernatant was collected and labelled as ‘wash’. 250µl of elution buffer was added to the washed beads for 5minutes at room temperature. The eluted GFP was collected after centrifugation. (3) Colloidal Coomasie blue stain (Bio-rad, Comassie Brilliant Blue G250) is used to determine the putative GFP.
Determination of Protein Concentration
Protein concentration was determined using the Bio-Rad Bradford Assay (1) (Bio-Rad Protein Assay Kit from United States) according to the the manufacturer instruction. Absorbance was measured at 595nm. A standard curve is plotted and the equation of the curve is y = 0.5147x + 0.0505 and with a R² = 0.9845. The concentration of BSA was 5mg/mL
Identification of GFP from transformed E.Coli cells by Western Transfer
SDS page gel was equilibrated in transfer buffer before being transferred onto Nitrocellulose membrane (NCF). NCF was blocked with blotto (5% skim milk with PBST) and incubated with primary antibody (Sigma Aldrich, Rabbit Anti-GFP) for 1 hour and with secondary antibody- enzyme conjugate (Sigma Aldrich, Goat Anti-GFP) for 1 hour. NBT/BCIP (Sigma Aldrich, Sigmafast BCIP(R)/NBT) is added for calorimetric detection of the protein
RESULTS
Figure 4: BSA concentration curve
Based on figure 4, with the given equation, the estimated Green Fluorescence Protein to have 0.602mg/mL. This concentration meant that in every 1mL, there would be a total protein of 0.602mg which included Green Fluorescent protein. The neat value (y-value): 0.361. This graph value had a R² of 0.984 which made the graph to be more reliable as the BSA concentration is nearer to the trend line.
Figure 5: Fluorescent Assay
In Figure 5, 259mg/mL of Green Fluorescent Protein. This would mean that in every mL, 259mg of Green Fluorescent Protein. It had a R² of 0.963 which an indicative that it the assay was reliable. (6)
Figure 2: PCR amplification of the GFP sequence sub clone from the pE-GFP vector into pQE30.
Figure 2: the result of all the lanes indicates that it has a high GFP concentration as an intense band is observed. This could have been due to PCR reagents being kept in proper conditions. The materials were probably being kept in an icebox as the result indicates an intense band, which resulted from the procedures to be done in the appropriate condition. Based on the result, a supercoiled band is observed. It also indicated that the the GFP was uncut that the PCR product does not have any contamination because only 1 band was observed. By comparing the product with the DNA ladder, the results show that the band has a base pair of approximately 750bp. Thick and supercoiled bands were observed for all the lanes.
Figure 1: Gel indicating restriction digests of Plasmid DNA for 3 individual teams.
Based in Figure 1, in lane 2 restriction enzymes were absent and served as control hence, one band was observed. the band appeared to be supercoiled and the band was thick. This was due to absence of restriction enzymes. It had 2000bp. Lane 3 had 2 bands, due to presence restriction enzymes. The band did not appear to be supercoiled as it is being digested with the restriction enzymes. The bands appeared to be linearized. It had 2500bp for the restriction enzymes whereas the positive green fluorescent protein had 750bp. In lane 4, it had 2500bp, supercoiled band is observed due to absence of restriction enzyme. In lane 5 it had 2 bands, restriction enzymes had a band of 2500bp and 1500bp for negative green fluorescent protein. This results corroborate with other 2 teams as their lanes corresponds with the above results.
Figure 3: Identification of GFP from transformed E.coli in western transfer with 35kDa.
Based on Figure 3, lane 2 appeared to have a linearized band and one band was observed. It served as an indicative that the Green Fluorescent protein was fully digested and no contaminates were found. The band appeared to be nicked, relaxed Circular. However, in lane 4 and 5, it appeared that it had 2 bands in each lane. It served as an indication that there are 2 types of protein in the lane. Supercoiled band were observed in lane 3 and 4.
DISCUSSION
Based on figure 4, there were scattering of points around the line drawn in the graph. The scattering was believed to be a feature function that determined the exact amount of protein present in the sample hence, the graph can be used to determine the protein concentration as the R² value was more that 0.9. This makes the result to be more reliable in determining the total protein concentration. Bradford Assay is beneficial to determine total number of protein and not specifically to green fluorescent protein. This Assay is less sensitive as compared to the fluorescent assay.(1)Based on figure 3, Ap-based detection system had to be done less that 10s as this would prevent background noise. This is because the dye would bind to the unbound protein and causing it to have multiple bands in a lane. This was because AP-based detection system was high in sensitivity which caused the reaction to happen quickly. Also, small amount of protein concentration would affect the rate of colouration as the binding will be faster. Conjugated antibodies increases the sensitivity of the assay. Also, use of monoclonal antibodies were more specific as it binds to the specific epitope.(7) In figure 1, controls were used to compare DNA digests with (lane and lane 4) and without enzyme (lane 3 and 5) which detects the changes that occurred independently of enzyme such as exonuclease contamination in the DNA or in any of the reaction components. (8) Low concentration of restriction enzymes would result to proteins to be cleaved inadequately. This would cause partial digestion hence, star activity would occur. High pH level would also affect the cleaving of the proteins. The ionic strength of the sample is to be done in a cautious manner as it would prevent multiple digestion or double digestion.(9) The figures were considered realisable as it met the parameters of the experiments and the protocol. Also, 3 teams shared similar results which concluded that the result were reliable. There would be increase in contamination when the enzymes or any other proteins were being shared amongst the group. Proper pipetting technique would .be beneficial to avoid cross-contamination in between the teams
References
1. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), pp.248-254.
2. Boffey, S.A., 1984. Agarose gel electrophoresis of DNA. Nucleic Acids, pp.43-50.
3. Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. and Stüber, D., 1988. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nature biotechnology, 6(11), pp.1321-1325.
4. Shi, H. and Su, W.W., 2001. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme and microbial technology, 28(1), pp.25-34.
5. Tsien, R.Y., 1998. The green fluorescent protein.
6. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall J. Biol. Chem, 193 (1951), pp. 265-275
7. Engvall, E. and Perlmann, P., 1972. Enzyme-linked immunosorbent assay, ELISA. The Journal of Immunology, 109(1), pp.129-135.
8. Hinds, K., Shamblott, M. and Litman, G. (1991) In: Methods in Nucleic Acid Research, Karam, J., Chao, L. and Warr, G. eds., CRC Press.
9. Robinson, C.R. and Sligar, S.G., 1993. Molecular recognition mediated by bound water: a mechanism for star activity of the restriction endonuclease EcoRI. Journal of molecular biology, 234(2), pp.302-306.
FIGURES AND FIGURE LEGENDS
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Figure 1: Gel indicating restriction digests of Plasmid DNA. 1st lane: Protein ladder,2nd lane: Control (+GFP plasmid with elution Buffer), 3rd lane: +GFP plasmid with BamHI and HindIII enzyme, 4th lane: Control (-GFP plasmid with elution Buffer), 5th lane: -GFP plasmid with BamHI and HindIII enzyme,
Figure 2: PCR amplification of the GFP sequence sub clone from the pE-GFP vector into pQE30. Each individual PCR sample was loaded into their assigned well.
Figure 3: Identification of GFP from transformed E.coli in western transfer with 35kDa.
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