Chris Lopez ’13: Dimensions Fellow in Pharmacology

On Sunday the membranes were cut into pieces and put into primary antibody and rocked overnight. Monday I washed these membranes and put them into secondary antibody. When these were done rocking I stained the AKAP1 bands with fast green. I then imaged the membranes.

I also ran eight SDS-PAGE gels. Four of the gels contained the input samples (the four tissues and their corresponding dilute samples). The other four were run like the ones last week; each tissue had its own gel with the NM samples. Once the gels were done running I transferred them onto nitrocellulose membranes. Afterwards I cut the membranes into three pieces; top, middle, and bottom. I stained the membranes with Ponceau S and imaged them before rocking them in primary antibody overnight.

Tuesday I put the membranes into secondary antibody and imaged them. I did band quantitation on the images and then I worked on the presentation I was to give on my project. The presentation is on Wednesday and I will present in front of the members of my lab.

Wednesday I presented to the members of my lab. I gave a powerpoint presentation and told them what my project was about. I also presented data and possible interpretations of what my results meant.

Thursday I made updates to my powerpoint and worked on the rest of the data I had to quantitate.

Friday I finished the quantitation on the data from the coomassie stained membranes. I made photocopies of all of my notes and printed all of the images I have scanned since the beginning of my project. I labeled all of the images and scanned them. I kept the notes and the images in a notebook for my use and made another notebook for the lab. I said goodbye to all of the lab members and thanked them for welcoming me into the lab and being great to work with.

The data I obtained was very interesting. I found that all of the tissues were able to pull-down PKA with the different regulatory subunits: RI, RIIalpha, and RIIβ. However, RI was bound much less. I also found that the ST-DE double mutation showed a significant decrease in regulatory subunit binding compared to the other samples.  This information could be important for future experiments. If the phosphomimetic mutations that I made have this much of an impact, then actual phosphorylation at the 315, 322, and 328 positions (S315, T322, T328) could have an even greater impact. If there is a decrease in PKA binding to AKAP1, then there would be a decrease in the amount of Drp1 that could be phosphorylated (PKA can phosphorylate Drp1). Drp1 is a protein that becomes active if it is dephosphorylated. In the dephosphorylated state Drp1 causes mitochondrial fragmentation through GTP hydrolysis. Therefore, if we can influence the amount of PKA that can bind to AKAP1 on the outer mitochondrial membrane, then we could influence the amount of Drp1 phosphorylation and therefore mitochondrial fragmentation.

My research pretty much laid the groundwork for other research. More research will be done to see the affect that actual phosphorylation at the phosphosites (as opposed to the pseudophosphorylation mutations I created) has on mitochondrial morphology.

Overall, this fellowship has been one of the greatest experiences I’ve had in my time at Cornell College. I have made great strides in my lab techniques and lab knowledge. I have also learned a tremendous amount about mitochondria and protein-protein interactions. I have met some great people in the lab and I am very grateful that I was given this opportunity through the Dimensions program at Cornell. This has shown me that hard work really does pay off and I accredit much of my work ethic to taking classes at Cornell. This research experience has given me a lot of insight as to what graduate school is like and what research is like as a career. I have only really considered going to medical school after graduating for Cornell College but I am now considering graduate school as an option as well. My experiences at Cornell and the classes I have taken there really prepared me for lab work at the University of Iowa. I will keep in touch with Stefan and Ron about what steps will be taken next in regards to the research and what is to come next.

Here are some pictures of my final week of research. A couple of them are during my presentation and another is when some of the lab members and I had a nice lunch outing at Short’s in downtown Iowa City (the burgers were delicious!).

Chris Lopez ’13: Dimensions Fellow in Pharmacology

Monday I ran four SDS-PAGE gels using the samples I made on Friday. I ran two gels for the rat brain samples and two for the liver samples. After the gels were done running I transferred them to a nitrocellulose membrane. I took the membrane and submerged it in dH₂O for 5 minutes before staining it with Ponceau S. I took a picture of it on the ScanMaker 6800 and then cut it into strips. I cut each blot into three pieces, the top, the bottom, and the middle. One middle portion of the rat brain blot was cut according to the calcineurin protein band and the middle portion for the second rat brain blot was cut according to the RIIβ protein band. The same was done for both liver blots. I blocked each membrane piece with BSA for 30 minutes. I then put the membrane pieces into primary antibody and rocked them overnight.

I also used the miniculture pellets (M ∆PKA) that were stored in the freezer last Friday to make minipreps. I used a miniprep protocol to isolate high quality plasmid DNA from E. coli cells. To the minipreps I added master mix (10X buffer 4, Bam Hi-Fi, Xho1, BSA, and dH₂O) to a small volume of the minipreps and incubated them for one hour. After incubation, I mixed the samples with 6X dye and ran them on an agarose gel using gel electrophoresis. I took pictures of the gel when it was done running. I chose the sample that looked best and mixed it with primer and water. I took the sample to a different lab so they could run PCR on it.

Tuesday I took the membrane pieces out of primary antibody and washed them with TTBS. I then put them into secondary antibody and let them rock for one hour. I washed them again with TTBS and imaged them on the Li-Cor Odyssey. I conducted band quantitation on the blots for RIIβ, RIIalpha, and calcineurin.

Wednesday I looked at the band quantitation data with Ron. It looked good but the mutations I made did not appear to have a significant effect on the pull-downs of the RIIβ and RIIalpha subunits of PKA. I will re-do the pull-downs and western blots to obtain more data and probably confirm my results. I will also probe for the RI subunit of PKA next time I do a western blot.

I did pull-downs for the seven NM samples using both the rat brain lysate and the liver lysate. I also made heart and kidney lysates to do pull-downs with. It will be interesting to see if the different tissues have different pull-down properties.

Thursday I ran two SDS-PAGE gels, one using rat brain lysate and the other using liver lysate. I ran these gels because I was going to check for the existence of RI subunit. I transferred the gels onto nitrocellulose membranes and stained them with Ponceau S before imaging them. I also used fast green on the AKAP1 bands. Fast green is a green stain that allows you to see protein bands. I blocked the membranes with BSA before putting them into primary antibody and rocked them overnight.

I also did pull-downs for the seven NM samples using heart lysate and kidney lysate. I put the samples into 2X sample buffer and stored them in the freezer overnight.

Friday I took the membrane pieces for the RI subunit (for brain lysate and liver lysate) and washed them. I put them into secondary antibody and let them rock before imaging them.

I also ran eight SDS-PAGE gels: two for the new brain lysate, two for the new liver lysate, two for the heart lysate, and two for the kidney lysate. I will check the samples for RIIβ, RIIalpha, and calcinerin. I will check the new brain and liver lysates, as well as the heart and kidney lysates for the RI subunit on Monday of next week.

After the gels were done running I transferred them to nitrocellulose membranes. I then stained them with Ponceau S. I let them dry out and stored them.

On Sunday Ron placed the membranes into primary antibody and let them rock overnight. On Monday I will probe the membranes with secondary antibody and image them.

This was a great week of research. I obtained results and I am repeating the experiments to obtain more results. Hopefully I will have all of the results by Wednesday and have the remaining few days for analysis purposes. Next week is my last week and I am excited to see what conclusions I can make based on my results.

Chris Lopez ’13, Dimensions Fellow in Pharmacology

On Monday I talked with Ron (who came back from vacation) about the results I had obtained. We took a look at the images of the nitrocellulose membranes for RIIalpha, RIIβ, calcineurin, and their corresponding GST-AKAP1 protein bands. The images didn’t look bad but they were a little messy. There was some background on the images and there were differences in the GST-AKAP1 bands, whereas they all should have been pretty similar. So Ron and I decided that I would go back and make tubes containing the same amount of 50:50 slurry mix, empty GST beads, and sample buffer that I used last Tuesday. I will not make brain lysate samples until tomorrow. We just want to check if the volumes we used of the slurry mix for the samples appear reasonable according to the protein concentrations that I determined. I ran the seven NM samples on an SDS-PAGE gel.

After the gels were done running I submerged them in water for five minutes. I poured out the water and added fixative solution, which contains acetic acid and methanol. The fixative helps attach the proteins to the acrylamide matrix. I poured out the fixative and added coomassie stain. I microwaved the gels for 12 seconds two times with the coomassie stain. This is a quicker way to stain the gels instead of rocking for one hour. I then poured out the coomassie stain and added fixative. I poured out the fixative and destained them before letting the gels rock for 10 minutes.

Tuesday I grew minicultures. I pipetted 5 mL of media (with ampicillin) into 12 tubes. I picked 7 colonies for the NM samples from ampicllin plates that I had kept and saved in the refrigerator.  I picked 5 colonies for the M samples; S315D, T322E, T328E, STDE, and Corrected. I incubated them in 10 mL tubes until I could visually see that cultures were growing. After the minicultures were done growing I grew 100 mL cultures.

Yesterday Ron made a PCR sample for ∆PKA for the M DNA, because I did not have one. I purified the PCR sample using a purification protocol. After the purification, I set up a digest for the purified DNA. I also set up a digest for a plasmid, which is what I will insert the ∆PKA DNA mutation into. To the plasmid and purified DNA, I added 10X buffer, two restriction endonucleases (BamHI and XhoI), and BSA. I incubated these two samples for 4 hours and kept in the fridge overnight.

In the meantime, I made more rat brain lysate because I will need more to do pull-downs. I chopped up some rat brain (1.6 oz) and transferred it into what is called a bouncing apparatus. To the apparatus I added 16 mL of Flag IP Buffer. I smashed the brain carefully and transferred the contents into a centrifuge tube. I centrifuged for one hour at 15,000 RPM. Once the mixture was done centrifuging, I poured out the supernatant and made 1 mL aliquots to use for the pull-downs. I stored the aliquots in the -80⁰C freezer.

I also made an agarose gel and saved it to be used tomorrow.

Wednesday I incubated the 100 mL cultures. I checked the optical density of the seven NM cultures on a spectrometer until the desired optical density was reached. When the cultures were ready I induced them with lactose and IPTG and incubated the flasks for another four hours. When they were done incubating I went through a process of centrifuging, adding buffers, and pouring off supernatant until I was left with cells pellets, which I stored for use on Thursday.

I also ran the gel from yesterday. I ran two M DNA samples (∆PKA uncut and ∆PKA digest) and two plasmid samples (uncut and digest). After the gel was run I cut out the ∆PKA digest bands. After this I used a gel extraction protocol to extract and purify the DNA from the agarose gel. I obtained the purified DNA from the gel. I didn’t use a gel extraction protocol for the plasmid DNA because I had leftover plasmid DNA from earlier on in my project. I checked the concentrations of the ∆PKA sample and the plasmid on a nanodrop. I added three different types of buffer to the two samples and let them incubate at room temperature for thirty minutes.

I grabbed DH5alpha cells from the -80⁰C freezer. I mixed the ∆PKA and the plasmid with DH5alpha cells. I heat shocked them and then “rescued” the tubes with plain media, which pretty much means after the heat shock I added 800 µL of plain media to the tubes. I shook them and incubated them for one hour. Then I swiped ampicillin plates with each sample.

Thursday I made a buffer consisting of Tris, NaCl, dH₂O, leupeptin, and PMSF. I mixed the cell pellets with the buffer and sonicated each conical for 30 seconds four times each. I centrifuged the conicals and poured out the supernatant. I went through a process of adding GST beads to columns and doing washes to obtain the seven NM protein samples in a 50:50 slurry of beads and buffer. I ran the seven samples on an SDS-PAGE gel with a marker and stained them with coomassie.

I also picked six colonies from the M ∆PKA ampicillin plate and grew minicultures.

Friday I imaged the SDS-PAGE gel on the Li-Cor Odyssey. I saved the images and conducted band quantitation to determine the protein concentrations of the seven NM samples. I then determined how much of the slurry mix and how much of the empty GST beads I would have to add to Flag IP Buffer in microcentrifuge tubes. I mixed the buffer with the beads in separate tubes. I went through a process of centrifuging, aspirating, and washing the tubes before adding rat brain lysate. I rotated the tubes for 90 minutes and went through another process of spinning, washing, and aspirating to obtain proteins. I added 2X sample buffer to the beads and heated them for one minute. I spun down the tubes and stored them in the freezer to run an SDS-PAGE gel on Monday.

I made liver lysate the same way I made brain lysate earlier this week. I repeated the same pull-down process with the liver lysate and stored those tubes in the freezer to run an SDS-PAGE gel as well on Monday. The reason for using liver lysate is because liver has a lot more AKAP1 protein than brain, so hopefully I will be able to pull down more protein using the liver lysate.

I also grabbed the minicultures from yesterday and centrifuged them. I poured off the supernatant and stored them in the freezer for use on Monday.

I only have two more weeks left and I still have to do pull-downs for the M samples, but I will be able to get that done next week. I also look to make much more progress on the NM samples so that my final week I can focus on analyzing my results. My lab techniques continue to improve and I hope to obtain significant results real soon!

Chris Lopez ’13, Dimensions Fellow in Pharmacology

This week I continued making progress on my project by doing pull-downs, western blots, and protein band quantification.

Tuesday I learned more about the purpose of my project. I knew that I was looking at the binding region of the PKA and AKAP1 proteins to see if the phosphorylation mutations I made would influence the binding of the two proteins. However, there is a protein called Drp1 (dynamin related protein 1) that hydrolyzes GTP and is involved in mitochondrial fragmentation. Drp1 is active in mitochondrial fission if it becomes dephosphorylated. Calcineurin can bind to PKA and can also dephosphorylate Drp1. So if the mutations I made appear to influence PKA binding to AKAP1, then the phosphorylation (or dephosphorylation) of Drp1 can be affected and therefore mitochondrial fragmentation can be affected as well.

Continuing with my project, I added different volumes of the sample slurry mix (GST beads and buffer) into new 1.5 mL tubes according to the concentration of the proteins, which was determined through band quantification last Friday. In other words, if a protein is more concentrated then I added less volume to a new tube, and if it was less concentrated I added more volume to a new tube. I wanted 40 µL total of beads but because the proteins had different concentrations I didn’t add 40µL of each sample slurry mix. To account for the remaining volume of beads, I added regular GST beads until the volume in each tube was 40 µL. To these tubes I added 200 µL of brain lysate, which is the source of the regulatory subunits of PKA (RI, RIIalpha, and RIIbeta). I put the tubes in a rotator to spin for two hours.

In the meantime I made a Flag IP buffer containing Tris, NaCl, Triton, leupeptin, Na₄P₂O₇, NaF, β-glycerol phosphate, and CaCl₂. I took the tubes off the rotator and centrifuged them at 4000 RPM for 2 minutes. I sucked off the supernatant using an aspirator and then added 500 µL of Flag buffer to each tube. I kept the tubes on ice. I washed the tubes with 1 mL of Flag buffer and pipetted the solution into new tubes. I repeated the process of centrifuging, aspirating, and adding buffer to the tubes. I spun the tubes a third time and sucked off the supernatant until about 30 µL of mixture remained in the tubes. To the tubes I added 10 µL of 4X sample buffer.

I prepared 3 brain lysate samples: one concentrated sample, one 50:50 sample diluted in dH₂O, and a 1:10 lysate sample diluted in dH₂O. I loaded the three brain lysate samples, the seven bead samples, and a broad-range marker onto an SDS-PAGE gel. I ran the gel for one hour. I transferred the gel onto a nitrocellulose membrane to be used on Wednesday.

Wednesday I continued working on the western blot, which is a method for detecting proteins. I took the nitrocellulose membrane, which now had the protein bands transferred from the gel, and I stained it with Ponceau S. I washed the membrane with water and scanned it on a Microtek ScanMaker 6800. This machine took images of the membrane with the protein bands. I exposed the gel to 2% BSA (bovine serum albumin). The purpose of the BSA is to bind to the “sticky” proteins on the membrane. In other words, the BSA blocks the non-specific interactions so that they will not bind to the primary antibody that will be used in the near future. I rocked the membrane submerged in the BSA for 45 minutes.

After the membrane had been rocked for 45 minutes, I cut out the portions of the membrane that are specific for the RIIA (R-two-alpha) subunit of PKA and the GST-AKAP1 fusion protein. I probed the two membrane portions with specific primary antibodies. I rocked these membrane pieces for three hours.

I took the membrane pieces from off the rocker and poured out the primary antibody. I washed the membrane pieces with TTBS (Triton Tris Buffer Saline) three times and then washed with dH₂O five times. I transferred the gels into conicals containing secondary antibodies. I covered these conicals with aluminum foil because they are light sensitive. The secondary antibodies are specific to the primary antibodies and they have fluorophores that will allow for the visualization of the protein bands. I rocked the conicals for three hours. I then carefully placed the membrane pieces into containers where they were submerged in 2% BSA and stored in the refrigerator overnight.

I ran two more SDS-PAGE gels. The gels will be given the same treatment as the one above, except that a piece will be cut out on one membrane for calcineurin (CaN), and the second membrane will have piece cut out for the RIIβ subunit of PKA. Also, pieces of the membrane will be cut out for the GST-AKAP1 proteins because the amount of fusion protein on each membrane is unique to each blot. After the two gels were done transferring onto nitrocellulose membranes, I repeated the same process I did for the first gel; staining with Ponceau S and washing with dH₂O, scanning, cutting protein bands, and blocking with BSA. I let the membrane rock in a refrigerator overnight.

On Thursday I took the membrane pieces that were in secondary antibody out of the conicals. I washed the membranes three times with TTBS and then with dH₂O. I imaged the pieces on a Li-Cor Odyssey machine.

I also took the membrane pieces out of BSA and cut the desired protein bands; one membrane for the calcineurin band and the other membrane for the RIIβ band, as well as pieces for the GST-AKAP1 proteins. I exposed the pieces to BSA (blocking) and rocked them for 45 minutes. After 45 minutes I took the membrane pieces out of BSA and exposed them to primary antibody. I rocked the pieces overnight.

I also conducted band quantification on the RIIalpha protein bands and GST-AKAP1 fusion protein bands. I conducted band quantification the same way I did last week. I made a powerpoint slide of the bands and band quantification data. I will use this powerpoint slide in a presentation I will do towards the end of my project.

On Friday I removed the membrane pieces from the primary antibodies and washed them with TTBS and then water. I made the secondary antibody mixtures that they would be put into. I added 15 mL of Li-Cor Odyssey blocking buffer into two individual conicals. I then spiked each conical with 1 µL of their respective secondary antibody. One conical was to be used for the RIIβ membrane (goat anti-mouse antibody at 680 nm) piece and the other conical was to be used for the two GST-AKAP1 membranes and the calcineurin membrane (goat anti-rabbit antibody at 800 nm).

I covered the conicals with aluminum foil because the secondary antibodies are light sensitive. I rocked the conicals for three hours. After three hours I took the membrane pieces out of secondary antibody and washed three times with TTBS. I then washed with dH­₂O before I imaged the membranes on the Li-Cor Odyssey, an infrared imaging system. I will conduct band quantification on the RIIβ, CaN, and corresponding GST-AKAP1 proteins and create a powerpoint slide for it, like I did for the RIIA and corresponding fusion protein yesterday.

Next week I plan to conduct a western blot for the RI subunit of PKA, and start the pull-down process for the M samples. I continue to learn more every day I am in the lab. I plan on moving through the M samples faster than I did for the NM samples because the procedures will already be familiar to me.

Chris Lopez ’13, Dimensions Fellow in Pharmacology

As I mentioned in my past blog, Stefan (the P.I.) and Ron (the post-doc) are out of the lab on vacation. So this week I worked with Andy, the M.D./PhD. student.

Over the weekend Andy grew minicultures in 5 mL conicals for the NM DNA samples. This week I will only be working with the NM samples because working on both the NM and M would be a lot of work, so I will save the M samples to work on next week.

Monday I grew midicultures out of the minicultures. I repeated the same midiculture process I used before for the four DNA mutations of NM, ∆PKA, and NM Not Corrected (NM without a mutation), into individual flasks. I checked the samples on the spectrophotometer to check the optical density. I checked at 600nm because that is the wavelength in which the E. coli cells absorb light. I continued checking until the desired optical density was achieved. I then added lactose and IPTG to the flasks and incubated for another four hours.

After incubating, the cultures were distributed into 50 mL conicals and centrifuged. The supernatant was discarded and the pellet was frozen for protein prep use tomorrow.

Tuesday I added 5 mL of a mixture containing, Tris, NaCl, Benzamidine, dH₂O, and PMSF to the conicals containing the frozen pellets obtained yesterday. I did the same treatment with an NM corrected sample for a total of 7 samples (∆PKA, the four mutations, NM not corrected, and NM corrected). I sonicated each sample for 30 seconds four times. Sonication pretty much bursts open the cells using sound energy, allowing for the desired protein to exit the cells. The mixtures in the conicals were transferred into large centrifuge tubes. The tubes were weighed to check for balance before they were centrifuged for one hour at 15,000 RPM.

Meanwhile, I added GST beads to lysis buffer in a 50 mL conical and let this mixture rotate for 1 hour.

After centrifugation, I grabbed seven columns, one for each sample. I then pipetted 15 µL of each centrifugation sample into small tubes for use on Thursday. I added the supernatants from the tubes into their corresponding column. I collected the flow-through and washed twice more with the flow-through. I then washed each column with lysis buffer on a vacuum manifold. I capped and topped the columns and shook them. I then added more lysis buffer to each column three times and made sure I washed all the beads into the liquid part of the column. I pipetted the liquid portion into separate 2 mL tubes, all the while keeping the samples on ice. I centrifuged the 2 mL tubes at 4000 RPM for three minutes. I poured off the supernatant leaving 0.5 mL of beads and buffer in the tube in a 50:50 slurry.

I did not due any lab work on Wednesday because it was the 4^th of July!

Thursday was a busy day. I ran the 28 samples I had on two gels using an SDS-PAGE technique. I also did a coomassie stain on both gels. SDS-PAGE stands for sodium dodecyl sulfate polyacrylamide gel electrophoresis. This is a technique that separates proteins based on their size. SDS is a detergent that can denature proteins and give them a negative charge, causing an even distribution of charge per unit mass and allowing the proteins to run in the gel.

I grabbed the 7 samples that were saved for use and that were not run on the column (supernatant samples) and added 4X sample buffer (containing SDS and other ingredients) to them.

I then grabbed the flow-throughs that were used on Tuesday and saved. I mixed each of the flow-throughs with 4X sample buffer.

I then grabbed the 2 mL tubes with the 50:50 slurry of beads and lysis buffer. I mixed the beads and the buffer by swirling and pipetting and pipetted 10 µL of each sample into new 1.5 mL tubes. With this I mixed 10 µL of 2X sample buffer, which was diluted from the 4X sample buffer. I now had 21 samples to run on the gel.

I pippetted 5 µL of the remaining slurry mix into new tubes and mixed with 45 µL of 1X sample buffer, which was diluted from 4X sample buffer. I now had a total of 28 samples to be run on the two SDS-PAGE gels.

I heated the samples (supernatant, flow-through, concentrated, dilute) for 5 minutes and then spun them down for a few seconds. I carefully transferred 10 µL of each sample into individual wells on the gels (each gel had 15 wells). I also ran the gel with a broad range marker, which is pretty much a mixture of proteins with different sizes that I will use to compare the size of the proteins in my samples. I used 1X running buffer in the box that the gels are placed into.

After running for an hour, I took the gels out and rinsed them with dH₂O. I placed in gels in a small box containing 30% methanol solution for 15 minutes. After 15 minutes I poured out the methanol solution and submerged the gels in a coomassie solution for one hour. After one hour I poured out the coomassie solution and submerged the gels in Destain solution. I let the gels rock overnight.

Friday I analyzed the gels I ran on Thursday. I ran both gels on a Li-Cor Odyssey machine, which pretty much digitizes and images the gels onto a computer through the use of lasers. I then used a program called ImageJ to analyze my gels, or more specifically the desired protein band. The gels looked good and so did the bands. There was some degradation which was to be expected, but the densest bands were the full-length proteins that I isolated. I determined the area under the curve, which just gives me an idea of which proteins are more concentrated or less concentrated.

Neither Stefan, Ron, nor Andy will be here Monday so I will wait until Tuesday to start the pull-down.

I am happy where I am at now with my progress; not only on my project but on my lab techniques and knowledge. I have learned a great deal in these past four weeks and I can only hope that I can learn even more in the next four. I look to make more progress next week, finishing the pull-down for the NM samples and doing the same for the M samples that will be started.

There are also a couple more pictures. The first one is when I was submerging the gel in Destain solution after the coumassie stain. The second photo is me sonicating the conicals with the E. coli cells in them.

Chris Lopez ’13, Dimensions Fellow in Pharmacology Research

This week went very well. I made a lot of progress on my project. On Monday I took the digests out of the freezer and added dye to all of them, including controls for the M and NM samples midiprep samples. Different master mixes with certain restriction enzymes were added to a small volume of the DNA digest samples. These were run on agarose gels using gel electrophoresis. Pictures were taken of these gels. A problem occurred, however. The controls run in the gel did not appear to be cut. The problem is that the control samples should have been cut by the restriction endonucleases in the master mix. To solve this problem, I added master mix to another small volume of the control samples that will be run on a gel tomorrow. As far as the rest of the digest on the gel, the DNA bands on the gels looked good.

The miniprep DNA samples were mixed with different master mixes, each with specific restriction endonucleases. These were left in an incubator to incubate overnight. Tomorrow they will be run on an agarose gel.  On Tuesday I did pretty much the same thing as Monday. I made five digests for each mutation for both the M and the NM DNA samples. I ran these digests on an agarose gel using gel electrophoresis. I also ran four controls in the agarose gels, two for the M midipreps and two for the NM midipreps. Finally, I reran the M and NM controls whose DNA bands did not appear to be cut on yesterday’s gels.  The gels worked well. I picked one sample from each mutation for both the M and NM DNA miniprep samples, and I mixed that DNA with primer and water. These tubes were taken to a separate lab so that linear amplification could be run on them.

 On Wednesday I didn’t do anything regarding my project, because I was waiting on the PCR results from the lab that I took my DNA samples to. The results usually take 1-2 days to come in. I am waiting on the results to see which samples I will choose to continue the experiment and do protein preps. So I just helped Ron (the post-doc I work with) with his project.

Ron’s project is similar to mine, except he is looking at another protein called calcineurin (CaN) and its interaction with PKA and AKAP1. He went through similar processes that I went through in the experiment and the next step in his experiment was doing protein preps. Helping Ron with the protein preps gave me the opportunity to learn how to run the experiment. I will be doing protein preps next week with the M and NM DNA samples.

Thursday I worked on both my project and helped Ron with his project. I will not discuss Ron’s project since it is not what I am working on. I will mention however, that I helped him with the pull-down assay, which is a technique I will be doing later on in my experiment after the protein preps.

As far as my project goes, I received the results from the PCR lab and the mutations (S315D, T322E, T328E, and the STDE double mutation) appeared to have worked, which was great news! This showed that I conducted the experiments up to this point correctly.  Proceeding, I used the minicultures I had made last week to grow midicultures (larger cultures) in flasks. I chose the minicultures according to which mutations (for M and NM) were tested in the PCR lab, since the mutations worked. I used the same process to grow midicultures that I used last week.  I then worked on another transformation. I transformed BL-21 E. coli cells from being ampicillin-sensitive to being ampicillin-resistant. After incubating for an hour, the eight samples (four mutations for both the M and NM samples) were loaded onto the ampicillin plates. The plates were left to incubate overnight.

Friday I did midipreps for the cultures that I grew in flasks overnight. I took the flasks out of the incubator and distributed each into two 50 mL conicals. I spun these down for 30 minutes and poured off the supernatant. I then used the Midiprep protocol with the vacuum method to obtain midiprep samples. This protocol is the same one I used last week to get midipreps. One other thing I did is pipette 400 µL of the DNA samples (before lysis buffer was added) into centrifuge tubes and added glycerol to them to make glycerol stocks. These stocks will be used in case the midipreps needed to be redone. I put the midipreps in the -80⁰C freezer when I was finished.

This upcoming week will be interesting. The P.I. and the post-doc I work with will be gone on vacation, so I will be left to do the experiments and interpret results mostly by myself. I will work on protein preps and pull-downs and if all goes well I should obtain the results for my project by the end of the week. The results will determine what I will work on for the next month.  I continue to learn new techniques and learn more about mitochondria and protein interactions almost every day. I really enjoy the lab I am in and the project that I am working on and like I said I look forward to getting results soon!

Today I will be doing midipreps for the cultures that I grew in flasks overnight. I took the flasks out of the incubator and distributed each into two 50 mL conical. I spun these down for 30 minutes. I poured off the supernatant. I then used the Midiprep protocol with the vacuum method to obtain the midiprep samples. This protocol is the same one I used last week to get midipreps. One other thing I did is pipette 400 µL of the DNA samples (before lysis buffer was added) into centrifuge tubes and added glycerol to them to make glycerol stocks. These stocks will be used in case the midipreps needed to be redone. I put the midipreps in the -80⁰C freezer when I was finished.

Chris Lopez ’13, Dimensions Fellow in Pharmacology Research

Here are some photos from the first week. I just figured out how to decrease the file size. The first one is me about to spread E. coli cells on an ampicillin plate. The second is me about to pipette the cells onto the ampicillin plate. The third is me with Ron Merrill, the postdoc I work with. The last one is me loading an agarose gel with DNA.

Chris Lopez ’13, Dimensions Fellow in Pharmacology Research

I made a lot of progress the second week of my fellowship. I learned more about the interaction between the AKAP1 and PKA proteins and more about lab techniques (digest, midipreps, minipreps, transformations, and site-directed mutagenesis). One of the best things about my second week is that I feel much more comfortable in the lab where I am working and I was able to conduct more of the experiments independently.

This week I will work on site-specific mutagenesis, also called site-directed mutagenesis. Site-specific mutagenesis is when a mutation is created at a specific site on a molecule of DNA. In my experiment, I will mutate a serine into an aspartic acid (S315D), and two threonines into glutamic acids (T322E, T328E) for both mouse samples. The human samples are assumed to have the same binding properties between AKAP1 and PKA. These will be silent mutations. The soon-to-be mutated serine and threonines are located in the AKAP1 protein binding site where PKA binds to AKAP1. It has been shown that the serine and threonines remain highly conserved and phosphorylated throughout the binding of the two proteins. If the mutation sites can change from being phosphorylated to dephosphorylated, then it could prevent the binding of PKA and AKAP1, which could have further downstream consequences.

Cultures were grown over the weekend using colonies on the ampicillin plates that I prepared last week. On Monday I started work on midipreps, which are very similar to the minipreps I did last week except on a larger cell. I was able to obtain the desired plasmid DNA. The concentrations of the samples were determined by nanodrop. The samples were put in a fridge overnight. I also made glycerol stocks with a glycerol solution and a small volume of the DNA samples in case the experiments needed to be redone at some point. After the midipreps were completed, a transformation was started, similar to the transformations conducted last week. The E. coli cells were transformed from being ampicillin-sensitive to ampicillin-resistant by inserting plasmid DNA into the bacteria cells. A plain SOC medium was added to the tubes containing the E. coli cells and plasmid DNA to allow the cells to start expressing the ampicillin resistance. The samples were allowed to incubate for one hour before they were placed on the ampicillin plates. The plates were allowed to incubate overnight.

On Tuesday, I made a mixture of the sample DNA, GST (glutathione S transferase) primer, and water for each DNA sample. I took these samples to a different lab that runs PCR for our lab. Linear amplification will be run on these samples.
The eight primers (oligos) that were ordered for site-directed mutagenesis came in today. These primers were diluted in Tris and mixed for ten minutes. The site-directed mutagenesis process was initiated using a site-directed mutagenesis protocol. The process is lengthy but the principle of the mutagenesis protocol is that there are complementary forward/reverse primers (for each DNA sample) harboring mutation and silent restriction sites that are used to synthesize entire plasmids (linear amplification) using proof-reading polymerase (Pfu Ultra). The template plasmids are destroyed by digestion with methylation-specific restriction endonuclease Dpn1. The template plasmids are methylated, allowing them to be destroyed by Dpn1. However, the newly created plasmids are created in vitro and are not methylated, so they cannot be destroyed by Dpn1.
The ampicillin plates were taken out of the incubator. Colonies from each plate were placed in separate tubes with media and were left in the incubator over night to grow cultures.

On Wednesday, I took the cultures out of the incubator and put them into flasks with media to grow larger cultures. The flasks were left to incubate for two hours before being checked in a spectrophotometer. The optical density of the NM (mouse) and H1 (human) cultures were favorable. However, the M (mouse) culture did not grow. This culture will be grown again another day. Lactose and IPTG were added to the NM and H1 samples and put back in the incubator overnight.
The DNA tubes were taken out of the incubator. A small volume of each was placed into new tubes to be run on an agarose gel. Dpn1 was added to the remaining DNA in the original tubes to destroy the template plasmids. These tubes were placed in an incubator.
I made an agarose gel and ran the DNA samples on it (including controls of the DNA samples that were not run in the PCR). The results were what we expected, with the DNA samples showing the desired band size and the samples that were not run on the PCR (for M and NM) showed no bands. No bands were expected for these samples because they did not have the mutation that was inserted into the DNA of the other E. coli cells.
Another transformation was done using DH5A E. coli cells. DH5A cells are better for expressing plasmids and DNA as opposed to BL-21 cells which are better for expressing proteins. I pipetted the bacteria cells into small tubes and added Dpn1 digest into each tube and allowed them to incubate overnight.

Thursday was an unusual day. The flasks containing the H1 and NM samples were taken out of the incubator and something unusual occurred. Inside the flasks were large chunks. This was not supposed to happen. What we expected to see was a culture of cells, but not large chunks. This may have occurred due to other bacteria already being in the flasks before the media and E. coli cells were added. This media could have out competed the E. coli cells and outgrew them. This may have been the reason why the cell culture containing the M DNA did not grow in the flask yesterday. The problem with this is the chunks in the flasks appear to be about the same quantity, so the likelihood of alien bacteria outgrowing the E. coli in each flask at about the same rate seems unlikely. Another possibility may be fungus contamination in the flasks. One more possibility for the chunks in the media could be that the E. coli bacteria overgrew and formed the chunks. Unfortunately this is a step back in my project and I will have to repeat growing the cultures.
The ampicillin plates containing the DPN1 digest samples were taken out of the incubator. Five colonies were chosen from each plate containing DNA with a specific mutation and placed into five separate tubes with media (forty tubes total). The tubes were placed in the incubator to grow over night.
I started work on the cultures that needed to be redone so I grabbed the original plates containing the H1, M, and NM colonies and placed one colony from each plate into separate tubes with media and ampicillin. These tubes were then placed in the 370C incubator to grow cultures.

Friday I took the cultures out of the incubator and placed the cultures into larger flasks to incubate. In two hours these flasks were taken out and checked on the spectrophotometer. The optical density of the cultures was right where I wanted it to be. Lactose and IPTG were added to the cultures and they were placed in a 370C incubator to incubate overnight and grow larger cultures.
The forty tubes containing E. coli cells with the mutation insert in the DNA were taken out of the incubator. They were centrifuged and the supernatant was discarded, except for one mL. The remaining supernatant was kept just in case the experiment needed to be redone. A miniprep was done on all forty cultures the same way I conducted minipreps last week.
Once the minipreps were done, a digest was done by making a master mix (containing restriction enzymes) and distributing an equal amount of master mix to forty clean tubes. A small volume of each DNA from each tube was pipetted into these new tubes. Six total controls were used using the Midiprep M and NM samples that were made on Monday. These will be used to compare and see if the digests contain the same insert that our Midiprep samples do. All these tubes were put in the incubator and left to incubate overnight. These samples are going to be run on an agarose gel and we hope to see that the digests have the same band size as the Midiprep samples. If the digests have a different band size, then it is possible that the DNA in the E. coli cells may contain unwanted insertions or deletions.

Once again the lab experience I have acquired through taking lab courses at Cornell continue to have a positive impact on my lab work during my fellowship. Not only that, but because of the intense workload and independence given in the lab courses, I have been able to adjust quickly to lab work at a different location outside of a classroom environment.

I have liked conducting research since the first lab course I took at Cornell, but now I am really becoming quite fond of it and I find it extremely interesting. I like knowing that what I am working on has the potential of having an impact on science and future projects.

Chris Lopez ’13, Dimensions Fellow in Pharmacology Research

I have had a great first week to start off my fellowship. I arrived at the main office in the pharmacology department in the science building I will be working in and introduced myself to the office workers, one of which was named Linda. Linda took me to the lab where I would be working and there I met Dr. Stefan Strack, the principal investigator for my research project. Dr. Strack is involved with many different projects going on simultaneously in his lab. Dr. Strack then introduced me to post-doc Ron Merrill and M.D./PhD. student Andy Slupe, the two main people I will be working with throughout the course of the fellowship. After being given a quick tour of the lab and being introduced to other researchers, along with being given my own desk and work space, I was given more insight into what exactly my project would entail of.

All of the projects going on in Dr. Strack’s lab have to do with mitochondria, ranging from mitochondrial fusion and fission to the influence of mitochondria in apoptosis. My project will focus more on the reguatory pathways of mitochondria and specific proteins that bind to mitochondria (PKA and AKAP1). I hope to better understand the interaction of PKA (Protein Kinase A) with AKAP1 (A Kinase Anchoring Protein 1) and how this regulates mitochondrial shape and function. It has been studied that mutations in proteins that bind to mitochondria during the regulatory process can lead to neurological disorders. Therefore, if we can gain a better understanding of these proteins and their interactions with mitochondria, we could theoretically come up with a basis for novel neuroprotective therapies in the long run.

My first day consisted of obtaining four DNA samples (two from mice and two from humans) and a vector plasmid sample. I ran the samples through a polymerase chain reaction (PCR) purification machine. Two restriction endonucleases or enzymes, BamH1 and Xhol, were added to the vector plasmid. These restriction endonucleases were used to cut a piece of the vector plasmid and remove it so that a portion of the DNA samples could be inserted. Phosphatase was then added to the plasmid sample to remove a phosphate group from the cut plasmid to avoid self-ligation.

My second day consisted of making an agarose gel. I added ethidium bromide to the gel because although ethidium bromide is a toxic and carcinogenic agent, this compound squeezes into DNA and fluoresces, allowing the DNA bands on the agarose gel to be viewed under UV light. I cut out the desired bands from the gel and treated them with a gel extraction method to obtain the desired DNA. I then ligated the DNA samples with ligase. The ligation samples were distributed into separate vials containing E. coli bacterial cells. These cells were treated in a way to allow the DNA (which was ampicillin-resistant) to enter the cells. The new cells were incubated overnight on plates with the expectation that the cells containing the ampicillin-resistant DNA would form colonies and the bacterial cells that do not contain the ampicillin-resistant DNA would not.

Wednesday was not as busy a day as the previous two days. I took the E. coli cells out of the incubator and the plates contained bacteria colonies as was expected. I then chose different colonies and placed them in a medium consisting of buffer and ampicillin. The ampicillin in the medium is important because it maintained the ampicillin environment, allowing for the bacterial cells with the ampicillin-resistant DNA to thrive. The samples were left to incubate overnight. This is the process I used to grow cultures.

The next day I used a plasmid miniprep protocol which was designed to isolate high quality plasmid DNA from E. coli cells. The process is a little lengthy but in short the cells were lysed with SDS detergent and the lysate was applied to a silica membrane column. The column was used to bind the desired DNA and the contaminants were removed. A mixture of restriction endonucleases, DNA, and buffer was made as part of the digest process. The DNA samples were added separately to a portion of this mixture. A digest is a way to prepare DNA for analysis. In this case, I took the purified DNA and cut it with enzymes to see which bacterial cells have the desired DNA insert.

Friday I made agarose gels and added the DNA samples to them. The samples were allowed to run and they showed the desired DNA bands. This showed that the restriction endonucleases cut at the right position. Next week I will work on site-specific mutagenesis. My first week of the fellowship was definitely a learning experience, and although I was not familiar with some of the techniques and procedures used, I feel like the lab experiences I have had in my science classes at Cornell, as well as the internship I had at the University of Iowa in March of this past school year, have helped prepare me for working in a lab environment. I have learned a lot my first week; information ranging from protein-mitochondria interactions to techniques new to me such as DNA purification and digest. I look forward to continuing my research here in Dr. Strack’s lab and becoming more comfortable with my project so that I can conduct more of the experiments independently. I also look forward to learning a lot more about mitochondria and proteins and their interactions, and I hope to obtain significant results in the near future.