Materials and Methods Growing the G Strain and Preparing the GCE (rGFP Crude Extract): To grow the bacterial culture, use 10 ml of liquid LB growth media for incubation. 500 ml of the bacterial culture is allowed to grow overnight at 37°C. It is later shaken vigorously to increase the OD600 to 0. 5, which means that time equals zero. At time zero, 1 mL of the culture is transferred into a 1. 5 mL centrifuge tube and centrifuged for 5-10 minutes to obtain a pellet. The supernatant should be discarded. The centrifuge with the bacterial pellet is labeled “GO” and stored at -20°C.
The culture is induced with 1 Mm of IPTG and allowed to keep growing. After 3 hours past induction, 1 mL of the culture is pelleted into a different 1. 5 mL centrifuge tube, and the bacterial pellet is labeled “G3. ” The centrifuge with Go bacterial strain needs 15 mL of the strain to be collected and to be pelleted into the centrifuge tube. The last pellet is labeled “G3-15 mL. ” Finally, both the G3 and G3-15 mL are to be stored at -20°C. Preparing the rGFP crude extract: 500 ul of breaking buffer (made up of 10 mM Tris, pH 8. 0 and 150 ml NaCl) is to be added twice to the G3-15 mL frozen bacterial pellet.
After adding breaking buffer, the pellet is to be immediately thawed and broken up by pipetting the breaking buffer up and down into the centrifuge tube. The homogenous solution of buffer and pellet is to be transferred into a 1. 5 mL centrifuge tube and vortexed for 5 minutes. The solution is to be centrifuged at 14,500 g for 10 minutes. The fluorescence seen in the pellet and supernatant samples are to be detected by using the hand-held UV light in the dark room, and are to be commented on for the relative levels of fluorescence. The supernatant is to be decanted to a new centrifuge tube without using the pipetman and labeled GCE.
Preparing a Ni2+-Agarose Column: Get a small amount of glass wool to put into a 3 ml plastic syringe and attach it to a ring stand. Students need to pipet enough breaking buffer into the syringe so it overflows. Work carefully to minimize the risk of air bubbles. Students can add more breaking buffer to the column and allow several drops to flow out to minimize the effect of the air bubbles. By the time there is at least 500 ul or 0. 5 mL of breaking buffer on top of the glass wool, 1 mL of 50% slurry of Ni2+-Agarose is to be pipetted into the column.
The luer-lock needs to be opened to pack the beads of Agarose matrix into the column. The column will be pre-equilibrated in meaning that 10 times more buffer than the bed-volume of the column is to be added to wash out ethanol left behind in the column. Loading the rGFP Sample into the Ni2+-Agarose Column: 100 ul of the GCE is to be transferred into a new centrifuge and set aside. The rest of the GCE is to be added to the column with the luer-lock closed. Then the luer-lock is to be opened and around 5 mL of the effluent is to be collected into a 1. mL centrifuge tube and label it W1. The column will continue to be washed by sequentially adding and collecting 0. 5 mL increments of breaking buffer. Each effluent from W1 to W10 must be carefully labeled and stored in trays. After collecting W10, the column is to be washed with 5 mL of breaking buffer. The final effluent is not to be collected in a centrifuge tube, but instead collected in the waste container. Eluting rGFP and Purifying Contaminants from the Ni2+-Agarose Column: Wash the column by sequentially adding and collecting 0. 5 mL increments of elution buffer.
Each effluent is to be labeled starting with E1 and concluding with E10. Determining Protein Concentration of GFP Fractions by using Bradford Assay: The goal is to generate a standard curve with six Bradford assays on the following amounts of BSA (Bovine Serum Albumin): 0, 2, 4, 6, 8, and 10 ug. Keep the solutions at room temperature for 10 minutes. Use the microplate reader is to measure the absorbance at 595 nm. The data collected needs to be plotted on a gridline-based graph and the standard curve is a line of best fit that is equally spaced in-between the highest and lowest data points.
The Bradford assay is performed three times in succession (in triplicate) for Wash 1-6 and Elution 1-6. The absorbance value will be extrapolated from the standard curve created on the gridline-based graph. The total amount of protein in ug that was present in the 0, 2, 4, 6, 8, and 10 ug samples’ volume of BSA is to be calculated. SDS-PAGE/ Coomassie Blue Analysis of rGFP Fractions: Students need to make a 5% and a 12% resolving gel by combining different quantities of water, 4x resolving buffer, 30% acrylamide, 10% APS, and TEMED using the pipetman set.
The students should construct an apparatus with two plates of glass: one thin plate, and one thick plate. The 12% resolving buffer will fill the majority of the apparatus, and the 5% stacking gel is only to be at the top with a plastic comb inserted to create lanes in the SDS-PAGE gel. The gel needs to be polymerized between the two glass plates until it is ready for use. After polymerization, place the gel into the electrophoresis tank after it has had the comb taken out, and has the tank filled with 1:10 diluted electrophoresis buffer.
Samples of GO, G3, GCE, E2, E4, W3, W5, and the ladder in the quantity dictated by the protocol’s table along with the specified amount of water, 4xSLB, and load onto the gel are to be added. They are to be vortexed for 1 minute, boiled for 2 minutes, and vortexed again for 1 minute. Lastly, they are to be centrifuged again at the maximum speed for 30 seconds. Then add them into the rows created in the electrophoresis gel and place in the electrophoresis tank for 45 minutes at 200 volts. After this, the gel can be taken out and stained and de-stained.
SDS-PAGE/Western Blot Transfer of rGFP Fractions: Add beta-mercaptoethanol to the 1. 5 ml centrifuge tubes (GO, G3, GCE, E2, E4, W3, and W5) and vortex for 1 minute, boil for 2 minutes, vortex again for 1 minute, centrifuge at maximum speed for 30 seconds, and then load the samples into another SDS-PAGE gel which is run for 45 minutes at 200 volts. The gel is then transferred into an container with 3 sheets of filter paper, nitrocellulose membrane, 12% resolving gel, and 3 more sheets of filter paper to enclose the gel were placed in order to preserve the gel.
Western Blot Development of rGFP Fractions: Use forceps to place the nitrocellulose membrane in the Tupperware container with the protein side facing up. A small triangle should have been removed from the gel to denote which side the protein was on. Ponceau S stain is to be added and kept with the shaking platform for ~1-2 minutes. Now rinse the nitrocellulose with deionized water and mark the MW ladder. Begin to remove the stain and add 5% non-fat dry milk/ TBS solution on the shaking platform for 30 minutes. The membrane is to be washed with 0. 05% Tween 20/TBS and left on the shaking platform for 5 minutes.
The washing and shaking process should be repeated two more times. The next step is the primary probing step, where the membrane is to be covered in 7 ml of mouse IgG anti-Xpress epitope MAb for 45 minutes on the shaking platform. The washing and shaking process should be repeated three more times and the second proving step is adding 7 ml of sheep IgG conjugated horse radish peroxidase polyclonal anti-serum solution. It is poured in and allowed to sink in for 45 minutes on a shaking platform. The sheep IgG washing and shaking process is to be repeated one more time.
The Tupperware container will be emptied and add 30 ml of TBS and shake for 5 minutes. 7 ml of TMB substrate solution should be added to the Tupperware container until the protein peaks are visible and at the right intensity. Add tap water to stop the development process. Dry the nitrocellulose membrane and take a picture as soon as possible. Results The E. coli strain BL21pLysS, PRSETA-GFP-uv represses the lac promoter which is part of the related genes that regulate other genes that are responsible for protein synthesis (the operon) for T7 polymerase activity.
T7 polymerase would bind to the T7 promoter of PRSETA-GFPuv and activate the His-6 tag that expresses GFP’s fluorescence. GFP is produced, but it is not enough for this experiment, so IPTG, a repressor, is added to increase the GFP production. GO is the first sample of E. coli. GO contained the GFP sequence while the others did not initially contain GFP. G3 was the next generation of E. coli after 3 hours of induction, W1-W10 were washes, and E1-E10 were elutions. Go initially had no IPTG, but was induced with the IPTG, so the G3 strain contained IPTG. The G3 strain’s IPTG led to an increase in GFP production.