Analysis of GFPS65T Protein - Assignment Example

Running head: Lab Report Lab Report [Writer’s Name] [Institution’s Name] Table of Contents 1. Introduction...................................................................................................................... a. Green Fluorescent Protein (GFP) b. Expression of proteins as Glutathione-S-transferase (GST) fusion proteins c. Statement of the Purpose d. Aim of this Experiment 2. Methods............................................................................................................................ a. Day 1 i. Setting up the PCR screen and streaking sampled colonies ii. Agarose gel electrophoresis of PCR products iii. Restriction endonuclease digests of pGEX-GFPS65T-1, 2 and 8 iv. Fluorescence scoring: GFPS65T-1, 2 and 8 b. Day 2 i. Agarose gel electrophoresis of restriction enzyme-digested plasmids ii. cDNA sequence analysis: pGEX-GFPS65T-1, 2 and 8 iii. Purification of GST-GFP iv. Fluorescence scoring: streaking E.Coli v. Collation and interpretation of individual and class data vi. Thrombolytic cleavage of GST-GFP vii. Collated class: PCR and fluorescence analysis E.coli viii. The size calculation of restriction digest products using the plasmid c. Day 3 i. Thrombolytic cleavage of GST-GFP (cont) ii. SDS-PAGE analysis of bacterial lysate, GST-GFP and GFP iii. Spectrophotometric measurement of GFP concentration (A280) using E iv. Bradford protein assay v. Coomassie blue protein staining vi. Calculation of the molar extinction coefficient for GFP using “protein calculator” vii. Spectrophotometric measurement of GFP concentration (A280) using E viii. Bradford protein assay 3. Results.............................................................................................................................. a. Day 1 b. Day 2 i. PCR and fluorescence analysis ii. Gel image of the PCR iii. Total yield of GFP from the purification process c. Day 4 i. RR for the non-induced sample ii. RR for the induced sample 4. Discussion........................................................................................................................ a. Day 1 & 2 b. Day 3 c. Day 4 i. CDNB assay for GST activity iii. RR for the induced sample 5. Summary of the Lab Work............................................................................................... Introduction In this practical the focus is upon one protein, that of GFPS65T. In this practical we are going to typify the expression vector pGEX-GFPS65T. This vector sets a fusion protein of GST and GFP-GFPS65T. Purification of this protein will then be done by affinity chromatography after which it will be cleaved using thrombin for the production of a pure preparation of GFP. Green Fluorescent Protein (GFP) The green fluorescent protein (GFP) is a protein made up of 238 amino acids. When blue light is shown on it, it displays bright green fluorescence (Prendergast & Mann, 1978). GFP gene is made use of in cell and molecular biology. In this field the use of this gene is as a reporter of expression (Philips, 2001). With a little modification, GFP has been used for making biosensors. Several animals have also been formed through which there is the proof-of-concept of GFP that there can be the expression of a gene in the whole organism. The introduction of GFP gene can be done in organisms and their maintenance follows in their genome. This maintenance is by means of breeding, injection using a viral vector, or through cell transformation. There has been the introduction and expression of the GFP gene in several bacteria, yeast and different fungi, fish, plant, and mammalian cells, which also includes human. In GFP the amino acids 65-67 have the florescent chromophore (Cheng, et al., 1996). These amino acids are serine-tyrosine-glycine in the case of the wild-type protein. A bright green light is emitted from the wild-type GFP which has λmax = 508nm. This emission takes place when the GFP has been excited with UV light (λmax = 395nm). However, in this case the excitation goes away soon with time (Cheng, et al., 1996). When the wild-type protein is excited with blue light (λmax = 470nm), it fluoresces stably, however, the fluorescence is weak. Because of such reasons there was a difficulty is detecting the wild-type GFP protein in plant and mammalian cells, and so there was not much application of them in these mechanisms. GFPS65T is a mutant protein. It contains just one base pair substitution of serine-65 to threonine (Heim, et al., 1995). This mutant protein has the most excitation at 490nm and the most emission at 511nm. When the E. coli-expressed GFPS65T is provided excitation with blue light there is it has six times more brightness as compared to the wild-type GFP. The mammalian cell-expressed GFPS65T is around 18 times brighter than wild-type GFP. The GFP being used in the practical would be a synthetic GFPS65T version that would have the favoured codons of very much expressed human proteins (Chui, et al., 1996). Expression of proteins as Glutathione-S-transferase (GST) fusion proteins The expression of foreign proteins is done as fusion proteins. This is owing to a couple of reasons. Since the protein is “foreign”, it has the vulnerability of degradation by the local proteases. However, when it is fused it is offered protection. Also, foreign proteins expressed in E. coli usually denature and precipate. The purification of GST fusion proteins is carried out using bacterial lysates by affinity chromatography by using immobolized glutathione. Fusion proteins are cleaved. After this the needed protein is eluted. Another method for eluting fusion proteins is by using mild, non-denaturing conditions by making use of reduced glutathione. Multiples cloning site is present on the pGEX vectors. Before this there is the sequence that encodes a thrombin or factor Xa cleavage site, a β-lactamase gene, a ptac promoter, and a laclq gene. This last gene is for constitutively directing the creation of the lac repressor which binds to the operator area of the tac promoter and thus it prevents expression. Statement of the Purpose Expression of the thrombin-cleavable fusion protein GST-GFPS65T. Aim of this Experiment In this experiment we are to ligate and transform the GFP. After which we have to pick a colony and perform PCR screening on it. Then it would be enzyme digested. Lysation would have to be done and then the purification of GFP-GST. Methods DAY 1 Setting up the PCR screen and streaking sampled colonies In the experiments the PCRs are supposed to be having a final volume of 20μL. The reactions have to be kept on ice at all times before the tubes are placed in the PCR machine. This step is necessary for the minimisation of there being the probability of the polymerase amplifying from oligonucleotides annealed non-specifically to the template. The amount of reagents which would be required for the PCR master mix was calculated. This calculation was done in the following manner: V1C1 = V2C2 Component One reaction μl/20μl X 8 Master mix μl/160μl Reaction buffer (10Xstock, 1Xfinal) 2 μl 16 μl dNTPs (10mM = 50Xstock, 0.2mM final) 0.4 μl 3.2 μl Forward primer (10μM=40Xstock, 0.25μM final) 0.5 μl 4 μl Reverse primer (10μM=40Xstock, 0.25μM final) 0.5 μl 4 μl Taq polymerase (5U/μl=12.5Xstock, 0.4U/μl final) 1.6 μl 12 μl Sterile MilliQ water (to 20μl final volume) 15 μl 120 μl Total 20 160 Labelling of 7 PCR tubes was done. There were 7 for each of the 6 colonies that had to be sampled and for a “template-free” control. Template free control was required for comparing the result we get from PCR tube. It is also used for detecting any contamination in tubes. After the labelling they were kept on ice. Following this a LB plate was taken, which had 100μg/ml ampicillin (it is used as a gene marker; cells that have ampicillin gene would survive in the media) plus also 0.25 mM IPTG. IPTG induces the transcription of gene coding for beta-glactosidase. Therefore, it is used for inducing gene expression. It would produce blue colour for colonies because of the presence of beta-galactosidase. The plate had been marked with our initials on its underside plus also the date was written there. Marking was also done of six sections for each of the six colonies which had been sampled. This is the side that has the agar as can be seen in Figure 1 below. Figure 1 – Organisation of PCR and streaking of sampled colonies In all the 7 tubes was added 20μl of PCR master mix solution after which these were kept on ice. The next step was the sampling of a colony for PCR. For the purpose of streaking on the LB plate there was the use made of a sterile yellow pipetted tip. For the sampling the tip was used for gently touching a colony after which it was quickly dunked into a PCR tube that had the PCR solution. Following this the same tip was used for streaking a sector from off the LB plate. The next step carried out was the placement of the inoculated plates in the labelled metal tin which had been provided. Next, there was the incubating done of the streaked colonies and the temperature for this was 37°C. This was done was a couple of hours after which the incubation was done overnight at 28°C. On day 2 this was observed for GFP expression under blue light. The PCR machines were preheated to 85°C after which the tubes were loaded into it. This preheating was done because the first cycle needs to start at 94°C and to save time, we preheated the PCR for around 15-20 minutes so that it would be easy to quick start from 85°C to 94°C. The following was the programming done for the PCR cycling. 1. 94°C 5 minutes 1 cycle (bacterial cell lysis and initial denaturation) 2. 94°C 30 seconds 3. 60°C 30 seconds X 35 cycles 4. 72°C 30 seconds 5. Hold at 4°C (until the amplified products are collected from the machine) Agarose gel electrophoresis of PCR products The solutions used which had been already prepared included the following: 1 X TBE buffer Gel loading buffer 0.089M Tris-borate 2mM EDTA pH 8.0 0.25% bromophenol blue 0.25% xylene cyanol FF 40% sucrose w/v in H2O 1mM EDTA Electrophoresis was done of the PCR products on 1% agarose gel (this had the SYBR-safe DNA stain). The electrophoresis was done in 1 X TBE buffer after which it was photographed. The next step was the printing of the images. Use was made of TBE buffer for the preparation of the gel and for filling the electrophoresis tank. 0.5g of agarose was added to 50ml of 1 X TBE Buffer in a 250 ml conical flask. These had been made totally molten in a microwave over after which it was cooled. 5μl of SYBR safe stain was taken. The agarose solution was then transferred to the gel tray that had been fitted with a 9-well comb. Next the well comb was removed gently and 5μl of DNA had been added to all the 20μl of PCR products. In the next step 20μl of this solution was loaded into the wells in the gel. 10μ (1kb ladder) 1 – 2 – 3 – 5μl of loading dye to each and load 20μl (PCR products) 4 – 5 – 6 – No template control 10μl lambda The gel had then been moved to the electrophoresis machine with 100V. Then it was removed and photographed under UV light. Restriction endonuclease digests of pGEX-GFPS65T-1, 2 and 8 The first step was the calculation for finding out the volumes of the master mix. The following illustrates the calculation: BamHI + Pstl digests Final conc. 1 reaction (20μL) Master mix (X3.5; 52.5 μL) 10XBamHI digestion buffer 1 X 2.0 X 3.5 7μL 100mg/mL BSA 1 mg/mL 0.2 X 3.5 0.7μL BamHI 20 U/μL 10 U/20μL 0.5 X 3.5 1.75μL Pstl 20 U/ μL 10 U/20 μL 0.5 X 3.5 1.75μL Plasmid template 100ng/ μL 5.0 - Sterile H2O 11.8 41.3 μL Total Vol: 20.0 μL 52.5 μL EcoRI + Pstl digests Final conc. 1 reaction (20μL) Master mix (X3.5; 52.5 μL) 10XEcoRI digestion buffer 1 X 2.0 7μL EcoRI 20 U/μL 10 U/20 μL 0.5 1.75μL Pstl 20 U/ μL 10 U/20μL 0.5 1.75μL Plasmid template 100ng/ μL 5.0 - Sterile H2O 12.0 42 μL Total Vol: 20.0 μL 52.5 μL Master mix (1) was prepared from the following: 1. 100mg/ml BSA 2. BamHI 20U/μl 3. Pstl 20U/μl 4. Plasmid template 5. Sterile H2O Master mix (2) was prepared from: 1. EcoRI + pstl digests 2. 10 X EcoRI digestion buffer 3. EcoRI 20U/μl 4. Pstl 20U/μl 5. Plasmid template 6. Sterile water 15μl of master mix (1) was poured into 3 tubes (which were all of size 1.5μl). after this 5μl (=500mg) was added to every plasmid pGEX-GFPS65T-1, 2 and 8. Then 15M of the master mix (2) was added to the rest of the three tubes after which was added 5μl of plasmid 1, 2 and 8. The zip spin was then used for mixing the contents. This was used in order that all the contents would settle at the bottom. The tubes were then placed in a rack which was labelled. The tubes were then incubated overnight. The temperature for this was 37°C. Fluorescence scoring: GFPS65T-1, 2 and 8 There was the provision made of these transformants for the purpose of scoring. The streaked colonies that were obtained had been then observed under blue light for fluorescence. GFP expression E. coli transformants give much stronger fluorescence at 28°C because it is the best temperature suited for GFPS65T for expression. However, it is not perfect at 37°C. DAY 2 Agarose gel electrophoresis of restriction enzyme-digested plasmids First the agarose gel was prepared and then the enzyme-digest was added (it was taken from day 1 lab preparation). Before this, however, had been added 5μl DNA loading dye. 1kb ladder (10 μl) Undigest plasmid pGEX-GFP-1 pGEX-GFP-2 EcoRI + pstl pGEX-GFP-8 pGEX-GFP-1 pGEX-GFP-2 BamHI/pstl pGEX-GFP-8 lambda 10μl The next step was placing the gel into electrophoresis machine and it was then run. After this the gel images were printed. cDNA sequence analysis: pGEX-GFPS65T-1, 2 and 8 The sequence that the tutor gave was used for analysing. Purification of GST-GFP To the lysate was added Triton-X such that the final concentration obtained is 1% (Triton-X is used as detergent to solublize the protein. It can be used in DNA extraction as a part of the lysis buffer). The stock solution used was 10%. After this it was mixed on a roller for 30 minutes and the temperature used for 4°C. The lysate was then transferred equally to some Eppendorf tubes which had a volume of 1.5mL. The next step was the taking of 100μL of supernatant for CDNB assay and stored. 25μl of supernatant was also taken for SDS-PAGE and stored. This was labelled with its name. The rest of it was stored in the 1ml glutathione agarose resin (10μl tube). Next mixing was done of it, and it was done through inversion. Next it was incubated for an hour on a roller at 4°C. In this time the protein was being binded to cell lysate (to the dye). Preparation was done of the BioRad and a beaker was placed under the column for collecting the effluent. The flow was checked by the addition of 0.5ml of PBS. The resin was then washed by adding 2 X 10 mL of PBS. After this was added 5ml of PBS-DDT (here the purpose of DDT is the protection that it provides on the GST groups. Also, it keeps it catalytically active). Next 1ml of PBS was added and the resin was resuspended gently. 25 μL of aliguot was taken for SDS-PAGE (it was labelled SDS-PAGE 2). Then 5μL of thrombin was added. Fluorescence scoring: streaking E.Coli Observation was made of the streaked colonies and after this they were scored under blue light for fluorescence. Collation and interpretation of individual and class data The results that had been obtained from the experiments were contributed to a composite table for the whole class. This was done during the lab. The table can be seen in the results section of lab book. Thrombolytic cleavage of GST-GFP 1ml of PBS was added to the column and then the resin was resuspended with care into the solution by pipetting up and down. After this 25μl of the sample of the 50% slurry had been taken for SDS-PAGE (the second one). This sample was then stored in the Eppendorf tube and the temperature used was -20°C. 5μl of thrombin was then added to the 50% slurry. Following the caping of the column and the mixing by means of inversion, incubation of the samples had been done for a night at room temperature. This was done on a rotating wheel. Collated class: PCR and fluorescence analysis E.coli Class of E.coli transformant PCR results Fluorescence? Number of colonies (x16) Whole class frequency Desired vector pGEX-GFP Yes: indicates that GFP contains S65T 6 5 6 5 6 5 33/36 = 92% Vector without insert pGEX-2T No 0 1 0 0 0 1 2/36 = 5.5% Vector with mutant GFP pGEX-GFP No: indicates S65T is not present or GFP contains another mutation 0 0 0 1 0 0 1/36 = 2.8% Other The size calculation of restriction digest products using the plasmid From this plasmid maps and from the PCR image we had got: pGEX-GFPS65T 1 (EcoRI + pstl) = (961 and 4703) bp pGEX-GFPS65T 2 (EcoRI + pstl) = (961 and 3987) bp pGEX-GFPS65T 8 (EcoRI + pstl) = (961 and 4703) bp pGEX-GFPS65T 1 (BamHI + pstl) = (1687 and 3977) bp pGEX-GFPS65T 1 (BamHI + pstl) = (971 and 3977) bp pGEX-GFPS65T 1 (BamHI + pstl) = (1687 and 3977) bp DAY 3 Thrombolytic cleavage of GST-GFP (cont) SDS-PAGE analysis of bacterial lysate, GST-GFP and GFP First of all assembling was done of the SDS-PAGE apparatus using a 12% per-cast polyacrylamide. Following this was done the preparation of 1:10 dilutions with PBS of samples SDS-1 and the non-induced sample that the control groups had provided (lanes 2+3 or 6+7 on gel loading scheme). The final volume of the dilutions was 25μ. Addition was done of 3-25μl of gel loading buffer to the diluted samples and also to the rest of the two 25μl SDS-PAGE samples that had been obtained on the second day. The next step was the incubation of the samples – incubation was done for 5 minutes at a temperature of 95°C. The samples were then placed in ice after which the following were loaded on our gel into lanes 15. Marker 10μl Non-induced lysate 10μl Induced lysate (SDS #1) 10μl Resin-bound fusion protein (SDS #2) 20μl Purified GFP (SDS #3) 20μl Following the loading of each of the 10 samples we connected the ledas to the gel apparatus and to the power supply (that was of 160-180 volts). We ran the gel for 60-90 minutes after which the dye-front reached the bottom of the gel. Spectrophotometric measurement of GFP concentration (A280) using E We set the spectrophotometric at 280 nm. Next we diluted our GFP (purified GFP). The dilution was 1:10 in distilled water (we had to make sure the total volume was enough such as to cover the light path in the curvette that was provided but should not be so much that we are wasting the sample). After this we set the spectrophotometer to zero by means of a solvent in which we had diluted the sample (distilled water). This led to the removal of all contribution to A280 that had been made by the components of the solvent. We measured the absorbance of the 1:10 dilution. We had to choose such a value that it ranged between 0.2 and 1.5. This was due to the fact that this is thought to be the most accurate range for the spectrophotometers made use of in the teaching labs. Lastly, we calculated the concentration by means of the Beer-Lambert law. An explanation is given for this in the lab book in the results of day 3. A280 = ECl Bradford protein assay We made 5 standards: BSA standards (μg/ml) 1000μl water 12 μl BSA + 988 μl water 20 μl BSA + 980 μl water 50 μl BSA + 950 μl water 75 μl BSA + 925 μl water 100 μl BSA + 900 μl water The following were our samples: 1:10 - 100μl GFP + 900μl water 1:50 - 20μl GFP + 980μl water Following this we added the following to the new tubes: 800μl sample + 200μl Bio Rad reagent. After this incubation was done for duration of 5 minutes after which we read the results at A592. Coomassie blue protein staining First of all we carefully transferred the gel to a staining tray. We covered the gel in coomassie blue stain (0.1% coomasie blue R-250 in fixatine (40% HeOH, 10% HOACl) covered with plastic wrap and incubated on a shaker for overnight at room temperature). Calculation of the molar extinction coefficient for GFP using “protein calculator” We performed the calculation of the protein proportional to light absorption by means of the Beer-Lambert law: A280 = ECI A280 = absorption at 280nm E = molar extinction coefficient for protein of interest C = concentration I = path length (the path of light passing a solution of the protein in cm). SDS-PAGE analysis Lane 1: Marker (kDa) Lane 2: SDS-PAGE #1 (induced lysate) Lane 3: SDS-PAGE #2 (resin-bound fusion protein) Lane 4: SDS-PAGE #3 (purified GFP) Lane 5: Non-induced lysate 4(a) GST-GFP 4(b) GFP- (26.9 kDa) Spectrophotometric measurement of GFP concentration (A280) using E A280 = E.C.I E=19890 C=?(m) L=1cm A280=0.128 A280 = E.C.I 0.128 = 19890 X C X 1 C = 0.128/19890 C = 6.43 X 10-6 μl 1M = mw(g)/L mw = 26.9 kDa 1M = 26.9g/L 1M = 26.9mg/ml C = 6.43 X 10-6 6.43 X 10-6M = x 1M = 26.9mg/ml 1M . x = 6.43 X 10-6 M X 26.9mg/ml x = 0.000172mg/ml x = 0.172Mg/ml = 172ng/ml 1:10 dilution (X10) 1720 ng/ml Bradford protein assay Concentration of BSA (Mg/ml) A595 tube A A595 tube B Average 0 0 0 0 1.2 0.045 0.079 0.062 2 0.102 0.119 0.111 5 0.222 0.238 0.23 7.5 0.297 0.323 0.31 10 0.355 0.392 0.374 1:10 GFP 0.314 0.239 0.277 1:50 GFP 0.172 0.168 0.17 Concentration of GFP from 1:10 dilution y = 0.0383x + 0.0277 (from the graph) 0.277 = 0.0383x + 0.0277 x = 6.51 Concentration of GFP = 6.51 X 10 = 65.1 Mg/ml Concentration of GFP from 1:50 dilution y = 0.0383x + 0.0277 (from the graph) 0.17 = 0.0383X + 0.0277 x = 3.715 Concentration of purified GFP = 3.715 X 50 = 185.8 Mg/ml Concentration of the purified GFP from (1:50 and 1:10) were different. This could have probably been owing to a mistaken reading or due to the difference in the condition of the substrate concentration. Results DAY 1 The clone PCR result Fluorescence Conclusion pGEX-GFPS65T-1 pGEX-GFP Yes This clone had a vector inserted with GFP pGEX-GFPS65T-2 pGEX-2T No This clone had a vector without insert pGEX-GFPS65T-8 pGEX-GFP No This clone had a vector with mutated GFP From the agarose gel picture we can clearly see that the lengths of each of the bands are of more than 868. This provides a proof for the fact that each of the colonies had been successfully transformed with pGEX-GFPS65T. However, we were unable to determine the concentration of DNA due to the fact that the samples had been over loaded (there is a requirement of diluting them). DAY 2 PCR and fluorescence analysis Clone number PCR result Fluorescence Conclusion 1 pGEX-GFP Yes All sampled colonies were successfully transformed with pGEX-GFP. GFP was successfully transformed into the plasmids. 2 pGEX-GFP Yes 3 pGEX-GFP Yes 4 pGEX-GFP Yes 5 pGEX-GFP Yes 6 pGEX-GFP Yes Gel image of the PCR Total yield of GFP from the purification process Method % GFP concentration A280 A280/CDNB 1.7/18500 X 100 = 0.0092% Bradford Bradford/CDNB 185.8/18500 X 100 = 1.004% We compared the amount of GFP fusion protein that had been initially present in the bacterial lysate with the amount of GFP recovered during the purification procedure (from A280 measurement as well as Bradford protein Assay). This was done for calculating the percentage recovery. This comparison resulted in the finding that the Bradford method is more of an effective manner of collecting more purified GFP as compared to the A280 method. DAY 4 The results for CDNB assay for GST activity: Time 0 0.5 1 2 3 4 Non-induced Induced 1:8 1 2 Average 0 0 0 0 0 0 0 0 0 0 0 1 0 0.01 0.024 0.081 0.087 0.098 0.032 0.284 0.201 0.242 2 0 0.021 0.044 0.153 0.17 0.196 0.057 0.579 0.463 0.521 3 0 0.033 0.064 0.219 0.243 0.29 0.086 0.826 0.646 0.736 4 0 0.044 0.084 0.281 0.314 0.374 0.115 1.04 0.873 0.947 5 0 0.056 0.102 0.337 0.380 0.454 0.143 0.170 1.03 1.1 Reaction rate (RR) RR = ΔAbs/Δtime RR at 0.5Mg/Ml concentration A340 at minute 3  y = (0.011 X 3) + 0 = 0.011 X 3 = 0.033 A340 at minute 1  y = (0.011 X 1) = 0.011 ΔA3.1 = 0.033 – 0.011 = 0.022 RR =0.022/3.1 = 0.011 RR at 1.0 Mg/Ml concentration A340 at minute 3  y = (0.02 X 3) + 0.002 = 0.062 A340 at minute 1  y = (0.02 X 1) + 0.002 = 0.022 ΔA3.1 = 0.062 – 0.022 = 0.04 RR =0.04/2 = 0.02 RR at 2 Mg/Ml concentration A340 at minute 3  y = (0.067 X 3) + 0.01 = 0.211 A340 at minute 1  y = (0.067 X 1) + 0.01 = 0.077 ΔA3.1 = 0.211 – 0.077 = 0.134 RR = 0.134/2 = 0.067 RR at 3 Mg/Ml concentration A340 at minute 3  y = (0.075 X 3) + 0.009 = 0.234 A340 at minute 1  y = (0.075 X 1) + 0.009 = 0.084 ΔA3.1 = 0.234-0.084 = 0.15 RR = 0.15/2 = 0.075 RR at 4 Mg/Ml concentration A340 at minute 3  y = (0.091 X 3) + 0.007 = 0.28 A340 at minute 1  y = (0.091 X 1) + 0.007 = 0.098 ΔA3.1 = 0.28-0.098 = 0.182 RR = 0.182/2 = 0.091 The standard curve for calculating the concentration of GST-GFP in induced and non-induced samples The change in abserbance/time in induced and non-induced samples RR for the non-induced sample: A340 at minute 3  y = (0.028 X 3) + 0.001 = 0.085 A340 at minute 1  y = (0.028 X 1) + 0.001 = 0.029 So, RR for the non-induced sample = (0.085-0.029)/2 = 0.028 RR for the induced sample: A340 at minute 3  y = (0.223 X 3) + 0.031 = 0.7 A340 at minute 1  y = (0.223 X 1) + 0.031 = 0.054 So, RR for the induced sample = (0.7-0.254)/0.223 = 2 When y = reaction rate (ΔA340/mn) and x = concentration (Mg/ml) Concentration of GST-GFP in non-induced lysate sample: y = 0.024x + 0.001 0.028 = 0.024x + 0.001 x = 1.125 Mg/40Ml Concentration of GST-GFP in induced lysate samples: y = 0.024x + 0.001 0.223 = 0.024x + 0.001 x = 9.25 Mg/40Ml X8(the dilation factor) x = 74Mg/40Ml For getting the concentration in MG/Ml 74/4 = 18.5 Mg/Ml For getting the concentration in Mg/Ml 18.5 X 103 = 18500 Mg/ml The final concentration of the GST-GFP = 185000Mg/ml Discussion Day 1 & 2 It was seen that the PCR sampled colonies gave 868 bp fragments in every colony. This refers to the statement that each of the colonies that had been sampled had been successfully transferred with plasmid which had GFP gene insert. Nevertheless, we were unable to estimate the concentration of the DNA due to the fact that the samples had been overloaded (there is a requirement of diluting the samples if the concentration is to be measured). Through the digestion by restriction enzyme it was confirmed the various sizes of fragments that had been attained through the inserted and uninserted plasmids Expected fragment size: BamHI and pstl = 971 + 3977 bp EcoRI and pstl = 961 + 3987 bp Expected fragment size: BamHI and pstl = 1687 + 3977 bp EcoRI and pstl = 961 + 4703 bp Application of digestion by restriction enzyme was done on three sample colonies (1, 2 and 8). The result was the transformation with plasmids that had various inserts. This confirmed the anticipated fragment size and observation under blue light (fluorescence). Through the fluorescence colonies it was proven that GFP had been successfully transformed into the plasmid due to the fact that each of them had the GFP fragment size. Fluorescence scoring of streaked E. coli plus GFPS65T-1, 2 and 8 transformants. It was found that the colonies in section 1 had been transformed with GFPS65T-1 under blue light while the colonies in section 2 and 8 were not. Another finding was that the colonies in section 8 had a vector without insertion with GFP. It was expected that the colonies in section 8 would fluoresce due to the fact that they had the GFP inserted in the vector. However, they did and for finding out the reason behind this we analysed the sequence for pGEX-GFPS65T-1 and 8. From the alignment it was seen that there was substitution on amino acid at position 66 from Threonine to Alanine and the result was non-fluorescence GFP protein in the section 8 colonies (pGEX-GFPS65T-8). The reason behind the 66 T/A substitution was the substitution of nucleotide at position 196 from Adenine to Guanine in pGEX-GFPS65T-8. Thus, there is a requirement of cross-check between morpholegcal appearance (fluorescence) and genotype for confirming whether the colonies had been transformed with uninserted plasmid or if the plasmids had mutant GFP. After we had compiled the groups data we found that 92% of transformation with plasmid had desired GFP gene while 5.5% of transformation with plasmid was without insert. From the result it was seen that there was high efficiency of cloning method applied for the GFP gene into pGEX-2T. Conclusion From the discussion above it can be concluded that each of our sampled colonies had been successfully transformed with plasmid that had GFP gene insert. It was also found that class frequency of transformation resulted in high efficiency of GFP gene insertion into pGEX-2T. DAY 3 In sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of bacterial lysate, GST-GFP and GFP experiment: the GST-GFP was purified and thromobolytic cleavage of GST-GFP was done. From the SDS-PAGE picture in lane 4(a), GST-GFP have molecular weight of ≈ 53.28kD as we expected. GFP in lane 4(b) is 26.9kD and that is normal as the molecular weight of GDP is 26995.23D. 3 important assays were made use of as quantitative assays in order to identify the concentration of GFP and GST-GFP. First of all, spectrophotometric measurement of GFP concentration (A280) was carried out using E. The abserbance at 280 for GFP was 0.218 with 1:10 dilution sample. It was quite low but we did not have extra sample for redoing it. The molar extinction coefficient for GFP is (19770 M-1 cm-1). By using the Beer-Lambert law: [A = E. C. I], we are easily able to carry out the calculation of concentration of GFP that had been around 6.43 X 10-6M. 1M GFP contained 26.9 mg/ml. Thus, 6.43 X 10-6 M had 1.72Mg/ml at 1:10 dilution. Second was the Bradford protein assay. The concentration of GFP had been 65.1Mg/ml with 1:10 dilution and 185.8Mg/ml with 1:50 dilution. 1:10 and 1:50 had different concentrations. This might have happened because of an error in spectrophotometer readings or because the condition of the substrate concentration had been different. The third assay was CDNB assay for GST activity. From this assay the concentration of GST-GFP was 18500Mg/ml. Thus, the calculation of the yield of GFP protein could be finally done. The answer was 0.0092% from (A280/CDNB) method and 1.004% from (Bradford/CDNB) method. Here the conclusion is that we were able to successfully purify the GFP protein and the Bradford method had been the best method that we used for getting the maximum concentration of the GFP. The percentage recovery of the efficiency of the purification procedure by means of the Bradford method was 1.004% and this is way too more than just 0.0092% by means of the A280 method. DAY 4 CDNB assay for GST activity We made up our CDNB standards. They are as follows: 0 0μl GST-GFP 40μl water 0.5 5μl GST-GFP 35μl water 1 10μl GST-GFP 30μl water 2 20μl GST-GFP 20μl water 3 30μl GST-GFP 10μl water 4 40μl GST-GFP 0μl water In duplicate: Preparation was done of our induced and non-induced CDNB samples and then 10μl lysate was added to 30μl water (1:4) X 2 In a yellow capped tube: (prepared CDNB assay solution) We took 8.8ml of distilled water, 10X reaction buffer (1ml), 100μl CDNB and 100μl of gluthatione solution. All these were caped and inverted many times. From the above solution was taken 960μl of it and poured into a standard cuvette and the reaction initiated when we added the 40μl standards (1ml total volume for each of the samples). We covered the parafilm with cuvette and then inverted it in order to mix it. Summary of the Lab Work PCR Amplified References Phillips, G. (2001). ‘Green fluorescent protein--a bright idea for the study of bacterial protein localization’, FEMS Microbiol Lett, 204(1): 9–18. Prendergast, F., & Mann, K. (1978). ‘Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea’, Biochemistry, 17(7): 3448–53. Read More