I can’t believe we only have two weeks left! The time has definitely gone by really fast!
This week was focused on figuring out if we actually made virus. The actual process takes a lot of time because we started making this during my first week and its now the 4th week. To do this we had to do a separation process and qPCR. I was very nervous because its not until the qPCR that we are able to see how much virus we actually made (also proves that we have been working with something for the past 3 weeks). Last week before our harvesting of cells we knew our cells had some virus in them since they also had the GFP gene (glowing cells). After harvesting it was time to lyse the cells and separate the virus from everything else (cell debris, cells DNA, RNA, etc). This process is done using Iodixonal tubes. You must layer the tubes using different concentrations of Iodixonal solution and after ultracentrifuging them, the virus concentrates itself in the 40% layer.
The process of layering the tubes was stressful because you start with the 15% and work your way to the higher percentages. Since you want to make sure you have clear layers you must go at a speed of 1ml per 10 seconds—- very very slow…After the layering and centrifuging, you must insert a needle on the top of the tube to let air out and a needle at the 40% layer to remove the virus. This was also very tough because you have to be careful not to go through and when removing the virus you also need to be careful to not absorb the white band above the 40% layer because that is more cell debris (you don’t want that). When this is done you are left with about 3ml of solution that has your virus– well, you are still not sure how much virus you have so qPCR is next.
For qPCR you must clean everything with bleach because you want to prevent your sample from being contaminated. We had to clean our sample a bit using NaOH and HCl, and some heat and then we put it in our wells. This process takes a bit of time because you have to be careful to micropipette just what you need. Some of the solutions you have to use are viscous and drops stick to the tip. You have to always pay attention and white any excess off because having an extra microliter can affect your reading. After micropipetting everything into the wells, these are taken to the qPCR machine which does all the work and gives you a lot of data after two and a half hours.
And….. the qPCR machine told us that we actually made 1.58E11 viral genomes per ml. My mentor said that this was a good result and our next step is to clean the sample further so it can then be used for Transduction. After 4 weeks of work I am so glad that we got some virus and it will be used for the research the lab is doing.
Week 3 went by really fast!
The first day of internship my mentor explained to me the sequence into making a virus and I didn’t realize that it would take so long! We are on our third week and we are still not done.
This whole week we have focused on the harvesting the cells portion and we still have more to go, but I got to actually see that the plasmid we have been making actually went into out HEK cells (human embryonic kidney cells). The way we know how our plasmid mad it in is due to a GFP gene that we insert into our DNA (some of your might have done the Transformation of E.coli with GFP lab, its kind of the same concept). After reading the protocols and watching a lot of You Tube videos about the prepping of the virus and the Harvesting process, I was really looking forward to seeing the cells glow– well, they don’t glow without a black light but with the blacklight they do glow!
Beautiful pellet with virus infected cells
Now that we are done harvesting and we have that beautiful green pellet at the bottom we have resuspended the cells and I got a chance to use Liquid Nitrogen to freeze them! I keep getting really excited about all things we are doing because its my first time doing it. I asked my mentor how many times he had created virus and his number is around a thousand!
Next week is Virus Purification and then we can run a qPCR to see how much virus we actually have!
In my lab we are working with a virus called AAV9. They are currently using as a vector for gene therapy, specifically to the heart. The virus is currently able to identify inflammation but the goal is to make it specific to a cell. To do this, my mentor is working with adding locks that will allow for the virus to only attach to a specific type of cell. This is very challenging and there are many steps to the process- from the creation of a virus with specific locks to the actual delivery of the virus- in vitro or in vivo.
My job in the lab currently involves keeping my HEK 293T cells alive- this involves splitting the cells: lifting them, changing them to another plate and adding new media to keep them happy. This is the one process that I feel very comfortable doing by myself and my mentor has trusted me to do it already about 3 times without his supervision.. yay! The rest of the time my mentor has been teaching me new protocols with the end goal of being able to make virus! Last week we started by preparing the backbone and the inserts that we wanted in our plasmid. Once the plasmid is ready, we utilize it to transform E. coli. We then send our E.coli samples to a company that is in charge of sequencing them and checks to see if the bacteria did indeed take in the plasmid. We sent in 8 samples and 4 out of the 8 were great! This allowed us to go on to what we have been working on this week- Maxi Prepping our DNA.
A Maxi Prep works as follows: Making large amounts of bacteria, spinning down the bacteria to a pellet –> lysing the bacteria to release DNA and protein –> precipitating the protein –> get rid of the protein and load DNA solution onto column that can capture DNA –> elute DNA and precipitate DNA –> pellet DNA and redissolve DNA.
What you see there inside the bottle is the pellet which is part of the first step in a Maxi Prep- I love pipetting but never have I had to spend a good 3 -5 minutes trying to re-suspend pellets– My thumb is definitely getting muscular! After that all the steps to follow involve cleaning the DNA because we want to make sure that we don’t have any cell debris or RNA in our final products. The pictures below show some of the cleaning/precipitation steps used in cleaning DNA. Picture on the left has a layer of gel, DNA and phenol/chloroform/isoamyl alcohol, After spinning them, the layers rearrange as the picture on the right with the layer of the phenol/chloroform/isoamyl alcohol on the bottom, gel and then the DNA on top which is what we will extract.
After all the cleaning and precipitation steps are done we will carry out a gel to see if we have clean DNA we can then use for our virus!
Its only Wednesday and I know I have been at my internship for only 3 days, but I am extremely glad to be here! This program is definitely not like other internships.
As teachers, we usually are the ones that instruct, teach and enhance the minds of students. For the past three days I have been the one learning, taking notes, asking questions, and learning new things! My mentor has been super patient and kind to me and has taken the time to explain the laboratory procedures (which they call protocols) that we will be doing throughout my time in the lab. There are definitely no dull or boring moments. I am either in the lab learning a new procedure or reading/learning more on the research we are helping accomplish.
Everybody at the lab is very helpful and everybody seems to help and learn from each other. This is great because it creates a great atmosphere to be around. I think its awesome that the graduate students at the lab are so willing to take people under them and teach them all about their research. I thought at the beginning that I would be the only “outside” person there, but there are many undergraduate students and high school students learning all the procedures and the importance of the research we are doing.
I am super excited for the weeks to come and at the same time nervous to be able to produce a lesson that relates to the research we are doing.