Today is the end of the second week of the REU program! I gained tons of valuable lab experience this week and started working more individually with less guidance. However, Clark was always there to supervise and answer any questions I had. The first two days involved doing IPTG induction and after that we analyzed the results using SDS-PAGE gels and western blotting.

Monday – IPTG Induction to Induce Protein Expression in E. coli

On Monday, I arrived at around 8:30 AM and we started off the day by making 4 liters of LB broth immediately. This is done, once again, by adding 25 g of LB powder and 1 L of Milli-Q water into an Erlenmeyer flask, mixing it using a magnetic stirrer, and autoclaving the mixture. Once the autoclave was done running (about an hour later), we cooled the flasks by putting them in a bucket with ice and water mixed together as water exchanges temperature faster than gases. Once the flasks cooled down and were safe to touch, we added 1 mL of chlorophyllin and 1 mL of carbenicillin to all four flasks. These are antibiotics and are typically added to cell culture media to prevent microbial contamination and grow select cells that have been engineered with the protein we are looking for. One important note is that all of this process must be done in a sterile field, which is the area immediately surrounding a Bunsen burner. Once the antibiotics were added, liquid culture of E. coli (two of 9337 and two of 10367) were added to each of the four flasks (10 mL per 1 L). Then, the flasks were placed in the environmental warm room to shake at 220 rpm at 37 degrees Celsius. They were shaking until they reached an optical density in the range of 0.5-0.6. The optical densities were periodically checked, the first time being after the 2 hour mark, by measuring the absorbance of the OD600 light using a spectrophotometer. Once they reached the ideal optical density, IPTG was added to each of the four flasks and they were placed in the 16 degrees Celsius shaking incubator overnight.

Tuesday – Protein Sample Extraction from E. coli

Tuesday started off with REU sessions until 12. At around 12:30 PM, I got to the lab and we immediately set to work, pelleting the colonies from yesterday on the centrifuge and pouring the excess supernatant into conical tubes. These were pelleted by centrifugation at 4000 x g for 10 minutes at 4 degrees Celsius. Then, the supernatant was poured off without disturbing the cell and the pellet was resuspended in sonication buffer. I noticed that as the cells were sonicated/lysed, the liquid became more viscous and less turbid. Following the lysis, the lysate was centrifuged at a high speed (10,000-20,000 x g) at the same temperature as before. Samples were taken of the pellets for both 10367 and 9337 as well as the supernatants. The next day, we planned on running SDS-PAGE gels, a process I had become familiar with last week, and Western Blots, a protocol that was completely new to me.

Wednesday – SDS PAGE and Western Blotting

On Wednesday, we immediately set to work at around 8:30 AM making 6 10% SDS-PAGE gels. First, we set up the apparatus by clipping two stacked gel plates to the casting frame and clipping 2 casting frames to each of the three casting stands. Once this was assembled, 1.5 mL microcentrifuge tubes were placed right between the clip on the casting stands to prevent leakage; this is not explicitly stated but is a trick Clark came up with after being in the lab for years. Then, DI water was added between each of the plates just to double heck for leakage. Once the apparatus was set up, the materials needed to make the 10% gel were compiled, but I will not list them here as they are easily accessible on a variety of protocols. Two different mixtures were made: the separating gel and the stacking gel. Under an applied electric field, the stacking gel concentrates the SDS-loaded linear protein molecules while the separating gel separates the proteins on the basis of molecular weight. The separating gel was made first, and all the ingredients were added at first except two: the TEMED and ammonium persulfate. This was because when these ingredients are added, the gel will begin to polymerize and must be immediately poured into the crevice between the glass plates. Thus, these components were only added when the gel was ready to be poured. Upon pouring the separating gel, the apparatus was placed into the incubator at 37 degrees to polymerize. Then, the stacking gel was made with all the same components except the pH of Tris-HCl used was 6.8 for the stacking gel rather than the 8.8 it was for the separating gel. Once the gels were made, SDS-PAGE was run for the samples collected yesterday (I’m not going to repeat how this was done as I mentioned it in last week’s blog post). Once the SDS-PAGE gel was run, Clark noticed a faint band he hadn’t seen before and we ran a western blot to confirm/support the results from the gel. Western blots are an analytical technique used to separate and identify proteins following SDS-PAGE. To run this blot, we prepared transfer buffer and assembled a transfer “sandwich” by soaking the gel and pads in the buffer. Then, to assemble the “sandwich,” we layered in the following order: sponge, pad, gel, PVDF paper, pad, sponge. Then, the proteins were transferred to the membrane in an apparatus that was placed in the fridge at 4 degrees Celsius at 100 V for one hour. Then membrane was then incubated in a 5% milk and TBST solution overnight in the environmental cold room, gently shaking at 4 degrees Celsius.

^ SDS PAGE gel

Thursday – Western Blot Analysis

Yesterday, there was a meeting until 10 AM and I reached the lab at about 10:20 AM. Then, we retrieved and analyzed the results of the western blot. We started by creating 5% milk and TBST solution and added primary antibody to it. Then, we poured this solution into the gel after washing it with TBST and let it incubate at room temperature for 1 hour with gentle shaking. Then, we washed the membrane with TBST three times for 10 minutes each to remove the primary antibody so the secondary antibody can bind to the protein. Then, we created another 5% milk and TBST solution and repeated the same process as we did for the primary antibody but with the secondary antibody. Once these washes were finished, we placed it in the incubator to let it dry and, upon taking it out, noticed bands for 10367 that Clark had never detected before. He will be continuing with molecular cloning for 10367 and I will now primarily be focusing on working with 9337. Once this was finished, we created LB broth and liquid colonies to incubate overnight (same thing we did on Sunday-Monday).

^ Western Blot

Friday – Repeat of IPTG Induction

Today, we repeated the same process we did on Monday with the IPTG induction (LB broth, colonies, antibiotics, IPTG, incubate). This marked the end of the week- a rather tedious week but fascinating and a valuable learning experience nonetheless.