05 Jul Liliana Almanza Mendez-Scripps Research @ Briney Lab, Week 2

We are back for my second week in the Briney Lab at Scripps Research, San Diego!

I’d have to say this week went by even faster than my first week and I have a feeling it’s just constantly going to speed up. This week was full of new wet lab experiences, which was so awesome. Oh and on anther note don’t worry I do not get starved at my house, I just eat a lot;)

Monday, June 29th

Terrance sent us the gene of interest sequence on HIV1 which we then had to copy into a website called Blast National Center for Biotechnology Information(ncbi) , this brings up all the known sequences on HIV1 and proves if its real DNA and to what viruses it matches with. My gene of interest ended best matching with HIV-1 Isolate Zm233M clone PB6 from Zambia. Terrance gave us a quick lecture on plasmids and how they tend to make mistakes, especially since bacteria is error prone. This means if a base changes —] the codon changes —] amino acid —] ending in an error produced protein. DNA is different because it goes through check points and usually if it finds an error it fixes itself and won’t proceed to the next step if all doesn’t look good. At 11 we went to a joint meeting with the Briney and Ward lab; were two students presented, one was on HIV and the other was on a article Stanford published about the new technologies used to analyze antigens. After the meeting we had to add the HIV-1 sequence with the HIV-1 isolate and match them up on Geneious and go through all of it and make sure there was no mistake, as far as I checked there was no mistake. Jonathan did a mini tour of the other floors in the building where he showed us all the machines lab’s had purchased but no longer use due to them being outdated or being too expensive to run. I kindly suggested having a yard sale but Jonathan said nobody would want them, I said really you don’t think anybody would want a DNA sequencer? I mean I would buy it if I had a couple thousand dollars to spare…

Any how we did end today pretty early so we walked over to USCD to take the trolley back home.

Tuesday, June 30th

Zoey’s mentor is back this means we get dropped off at the lab at 7am. This gives Belle, Ellie and I two hours of free time before we start. So Jennie took us to a cafe then dropped us off at Torrey Pines (a beach) that’s only like a 20 minute walk from our lab. We started the day by making 10L of media which if you don’t recall is like cell food and contains NACL, Bacto Yeast Extract, Bacto Tryptone and water. We had to be cautious considering we were over filling the bucket where we were mixing all of the ingredients in. After making the media which smells like broth/dog food we had to transfer it to multiple flasks, after packaging we put them inside the Autoclave which is basically a pressure cooker, this makes sure the media is sterile. Meanwhile we waited we started Gibson assembly with the sample of Hanta Virus and HIV DNA. To begin we had to resuspended the DNA since both were in powdered form, we put them in a centrifuge tube where we added to virus samples. After a minute we added  the vector (SCT backbone) and finally our gene of interest. Once we centrifuged it all down to the bottom of the tube we went over to the ThermoCycler, its takes the samples and keeps them at a steady temperature. Then, we went through the second part of the procedure which is heat shock transformation, we took our samples and heated and cooled them, by giving them ice baths or inserting them in the ThermoCycler. Why at different temperatures? Well each enzyme inside the Gibson Assembly activates at different temperatures thus the changes in temperatures, also by doing this it makes the membrane become more permeable which allows organelles to flow in and out, which is the purpose of Gibson Assembly. We now wait half an hour for our samples on the shaker plate, which also keeps them at a constant temperature. After they were done we began transfection onto the agar plates which we streaked using glass beads. Finally we stored them in the warmer fridge with the culture media, hopefully tomorrow morning we can see colonies formed. This can all demonstrate how clean our transfection technique is… After work I went on my usually run then to adoration and got a really cute wax blue rosary:)

Wednesday, July 1st

Well I encountered my Count #1 around 10am when Jonathan showed us the agar plates from yesterday, in which showed no colonies but a ton of air bubbles. Count #2 occurred when we found out 7L of the media we made yesterday was unusable because bacteria was growing inside already. Turns out it wasn’t our fault but the Autoclave that didn’t reach a high enough temperature or the media wasn’t inside long enough.  Terrance suggested we just start over again so I began washing all the beakers, Belle started making labels and Ellie began making the media. We were definitely more efficient and indeed Jonathan checked the tape was black before we emptied the AutoClave. Afterwards Nathan invited us the microscopic he had used last week, so we followed like ducklings. Nathan first had to add a radioactive solution to make the sample antigens visible underneath the scope. We then went over to a ”vacuum” oriented machine that spins the plasmid, I got to insert the sample and press go (SCIENCE)!! When running the machine looks like a galaxy with a purple background and stars floating. Today Nathan was looking at some Ebola samples, they were very good samples and showed antigens binding regardless of not following the recommended storage procedure. Since Nathan saw antigens bind he will go ahead and process it to an algorithm to turn it into a 3-D structure for further research. This is also where we meet Nate a 1st year grad student, he was so glad he was no longer the youngest in the lab. Afterwards we headed back to the Briney lab where Nate explained his project, which started when he traveled to Sierra Leone with Jonathan and Benjamin. They got to analyze many blood samples of affected and non-affected people who had Ebola and other diseases in the same family. They are now all just waiting to receive the samples to do further studies here in San Diego. Meanwhile Nate waits he is designing antigens that could bind to multiple viruses within the same family.

Thursday, July 2nd

Belle and I arrived at the campus earlier than needed so we hung out by a cute outdoor fountain area and drank a delicious white mocha coffee. Jonathan led us through a protein purification which was a very long procedure. To begin he brought out the two sample viruses and I was assigned CTLA-4. CTLA-4  is an antibody against cancer, it binds to the receptor the bad guy and waits for a signal saying ”shoot the bad guy” to deactivate it. So first we resuspended the beads (virus) inside the samples or the precipitate that had formed at the bottom of the tube. Then, we labeled 24 1.5mL centrifuge tubes, the filters went on top. So this process wouldn’t take so long Jonathan showed us how to use the multi channel pipette’s, which speed up the process. Since this was Affinity chromatography the filter was built so only the antibodies stayed in the filter and all the protein and junk followed to the bottom. After we centrifuged two times we added 1 round of 1xDPBS   (buffer). Lastly, we added three rounds of IgG buffer,  these three runs of buffer were to ensure no debris was caught up in the filters. After all the pipetting my fingers were a little tired and my hands began to get shaky, but I was just beginning difficult because Jonathan says Terrance at the time was doing 96 samples!! Turns out none of the samples had adequate concentrations to continue or qualify for the next round of testing.  Jonathan predicts it was the wrong reagent, so we will test the theory when the new samples come in…

Friday, July 3rd

Today we didn’t go to the lab, so we invited Izzy an intern at UCSD over to the house. We all decided to head to Mission Beach, but we had some complications parking due to the 4th so we just dropped off Belle, Zoey, Kalia, and Izzy. Jennie got home and we headed over to Costco which was packed as expected but we did secure all the good snacks, like chocolate crepes and 2 tubs of Açaí!!

Saturday, July 4th

Happy 4th of July!! Today was a very easy soft day which started with me going to mass. After mass I walked home but as I wasn’t paying much attention to the directions I had inserted in my phone, I ended up turning a one hour walk into two hours! Which resulted in me getting a very bad fanny pack tan line. As a house we decided to spend our afternoon painting, hence the toad and the fingerprints, although I do have to wait for Ellie to get back home to add hers. In the process we had two amazing charcuterie boards to fuel all of our creative juices. We ended the night by watching the fireworks at the Catholic Dioceses which is about a 8 minute walk from the house.

See you all in week three:)