18 August 2010: The Final Post
Well, we have come to the last post of my summer research blog. Sigh. I cannot believe I am already finished in the lab--time has passed SO quickly. I feel like I have passed very quickly from knowing very little about telomeres (last winter), to reading everything I could on them and becoming fascinated but not actually expecting my own project (later last winter), to being a little freaked out but totally excited to design and take on my own project in the lab (beginning of this summer) to finally preparing all my results in a paper to publish (now!...a final version coming soon to this blog :] )
The process that began last winter and is now wrapping up has provided me a diversity of new experiences, but as I reflect on the whole thing, one thread seems common to the whole experience: the necessity to ask questions.
If I have done anything important along this research process, it has been to ask questions. First, questions to myself: What do I want to do with my summer? Are there any research labs in Spokane? Then, questions to others: Can I come work in your lab? Will you grant me the financial resources to make this experience possible? Eventually, questions that could only be answered on the bench: Will a certain mutation cause telomeres to elongate? Why are we seeing a mutation more frequently in cancer cells than in non-cancerous ones? Finally, I hope these questions will find answers that will feed the larger questions that are being tackled all over the world: What do these results mean for the overall picture of telomere replication? How can we take translate these findings into cancer therapies?
Although answering questions is, intrinsically, the ultimate goal of a research process, I find that I am more interested in the future questions than in the results that have been generated by my experience. For those reading this blog who have not had a research experience themselves: ask some questions yourself! The expanse of what we have to learn even just about telomeres is so vast, my mind is boggled by what we have to explore in the wider fields of cancer, biology, and science holistically. There is definitely a place and a need for all inquiring minds to undertake research projects in any field of interest: all you have to do is ask!
While I will be posting my completed paper once it comes to publishing, for now, this is the final post.
Thanks for reading about my research quest this summer, and I wish any future researchers all the best in their inquiries.
So long!
The process that began last winter and is now wrapping up has provided me a diversity of new experiences, but as I reflect on the whole thing, one thread seems common to the whole experience: the necessity to ask questions.
If I have done anything important along this research process, it has been to ask questions. First, questions to myself: What do I want to do with my summer? Are there any research labs in Spokane? Then, questions to others: Can I come work in your lab? Will you grant me the financial resources to make this experience possible? Eventually, questions that could only be answered on the bench: Will a certain mutation cause telomeres to elongate? Why are we seeing a mutation more frequently in cancer cells than in non-cancerous ones? Finally, I hope these questions will find answers that will feed the larger questions that are being tackled all over the world: What do these results mean for the overall picture of telomere replication? How can we take translate these findings into cancer therapies?
Although answering questions is, intrinsically, the ultimate goal of a research process, I find that I am more interested in the future questions than in the results that have been generated by my experience. For those reading this blog who have not had a research experience themselves: ask some questions yourself! The expanse of what we have to learn even just about telomeres is so vast, my mind is boggled by what we have to explore in the wider fields of cancer, biology, and science holistically. There is definitely a place and a need for all inquiring minds to undertake research projects in any field of interest: all you have to do is ask!
While I will be posting my completed paper once it comes to publishing, for now, this is the final post.
Thanks for reading about my research quest this summer, and I wish any future researchers all the best in their inquiries.
So long!
28 July 2010: A Day in the Life
A Day In The Life
“Wake up in the morning, feelin’ like P. Diddy...” But no, really.
By some stroke of amazing good luck, a day in the lab for me starts...whenever I feel like starting. This is ususally around 8 or 8:30 A.M. for me, but the rest of the lab generally won’t trickle in until 10 or so. I like the quiet mornings when I am the only one in the lab—it gives me time to get all set up for the day, concentrate, and get to the tissue culture hood before anyone else!
When I first get to work, I usually have some type of cells to whom I must pay a visit. This morning, I first took seven bacterial plates out of the incubator to check their growth—I preformed a transformation of these bacteria yesterday, and today I am inspecting the colonies that have grown on the plates.
After checking my bacteria, I head to the tissue culture room and inspect my dishes of cancer cells under the light microscope. I check to see that the cells are happily growing, not too full, not too dead, and moving in the right direction (either gradually filling up their dishes if I am simply growing them, or gradually dying if I am trying to select cells with a certain drug resistance). Tissue culture is usually the first procedure I will do in the lab.
Tissue culture is an art as much as it is a science and requires very sterile technique. It involves changing the media in which cells live, splitting cells from one dish into several dishes, collecting cells to freeze or isolate DNA, and adding drugs to the cells. I do TC first because sometimes, I will split cells in the morning and add selecting drugs in the afternoon, or preform other tasks in a time-sensitive manner.
After I work on the cells I am culturing, I will return to my bench to work on other experiments. For example, I might run a Western Blot one day to check whether cells are expressing a target gene. Running a Western involves mixing buffers, casting gels and letting them sit, mixing samples of DNA and dye, loading the samples onto the gel, letting the gel run, sandwiching the gel between paper and membrane to transfer the gel contents to the membrane, running a current through the gel-membrane contraption, and then placing the membrane on a rocker with different chemicals and antibodies to let it “develop.” Many of these steps (running the gel and the gel-membrane) take an hour to run, so I will plug the gel box in and then go do something else. This could include running the autoclave (a large machine that sterilizes things we use in the lab, like pipette tips and microtubes, with super-he ated steam and pressure) or grabbing lunch.
Additionally, I often prepare PCR reactions while Westerns run. This involves mixing DNA and chemicals in a tiny, tiny tube (they look like they were made for barbie dolls!), and then designing a program on a thermocycler machine to run the PCR. Many times, PCR will take 8 hours or so to run, so I will leave the machine running over night and collect my samples in the morning.
All in all, I am usually in the lab until 5 or 6 in the evening, although there are also many days where I leave by 3. Although days in the lab vary a lot depending on when cells become ready for different procedures, the day usually flies by no matter what I'm doing!
“Wake up in the morning, feelin’ like P. Diddy...” But no, really.
By some stroke of amazing good luck, a day in the lab for me starts...whenever I feel like starting. This is ususally around 8 or 8:30 A.M. for me, but the rest of the lab generally won’t trickle in until 10 or so. I like the quiet mornings when I am the only one in the lab—it gives me time to get all set up for the day, concentrate, and get to the tissue culture hood before anyone else!
When I first get to work, I usually have some type of cells to whom I must pay a visit. This morning, I first took seven bacterial plates out of the incubator to check their growth—I preformed a transformation of these bacteria yesterday, and today I am inspecting the colonies that have grown on the plates.
After checking my bacteria, I head to the tissue culture room and inspect my dishes of cancer cells under the light microscope. I check to see that the cells are happily growing, not too full, not too dead, and moving in the right direction (either gradually filling up their dishes if I am simply growing them, or gradually dying if I am trying to select cells with a certain drug resistance). Tissue culture is usually the first procedure I will do in the lab.
Tissue culture is an art as much as it is a science and requires very sterile technique. It involves changing the media in which cells live, splitting cells from one dish into several dishes, collecting cells to freeze or isolate DNA, and adding drugs to the cells. I do TC first because sometimes, I will split cells in the morning and add selecting drugs in the afternoon, or preform other tasks in a time-sensitive manner.
After I work on the cells I am culturing, I will return to my bench to work on other experiments. For example, I might run a Western Blot one day to check whether cells are expressing a target gene. Running a Western involves mixing buffers, casting gels and letting them sit, mixing samples of DNA and dye, loading the samples onto the gel, letting the gel run, sandwiching the gel between paper and membrane to transfer the gel contents to the membrane, running a current through the gel-membrane contraption, and then placing the membrane on a rocker with different chemicals and antibodies to let it “develop.” Many of these steps (running the gel and the gel-membrane) take an hour to run, so I will plug the gel box in and then go do something else. This could include running the autoclave (a large machine that sterilizes things we use in the lab, like pipette tips and microtubes, with super-he ated steam and pressure) or grabbing lunch.
Additionally, I often prepare PCR reactions while Westerns run. This involves mixing DNA and chemicals in a tiny, tiny tube (they look like they were made for barbie dolls!), and then designing a program on a thermocycler machine to run the PCR. Many times, PCR will take 8 hours or so to run, so I will leave the machine running over night and collect my samples in the morning.
All in all, I am usually in the lab until 5 or 6 in the evening, although there are also many days where I leave by 3. Although days in the lab vary a lot depending on when cells become ready for different procedures, the day usually flies by no matter what I'm doing!
23 July 2010
To depart from the postings of methodology and results I have been writing, I thought today I'd include a short update on some of the non-research aspects of my lab!
First, a brief introduction to the other members of my lab. Our lab has operated with five members for most of the summer, but we have a new lab tech who has just joined our group this week. The Primary Investigator in my lab is Dr. Chai, who is a faculty member of the WWAMI program and Washington State University's School of Molecular Biosciences. In our lab are also two post-docs, Dr. Dai and Dr. Huang, who were recently listed as the first authors on this paper (a very interesting one in the telomere field!) The other full-time member of the lab is Shilpa S., who is a Ph.D. student originally from Bangalore, India. All of the lab members have been a great help to me as I have learned to culture tissue, run different reactions, and navigate my way around the lab--I have been very fortunate that all the members of my lab are outstanding teachers. Now, I am playing a bit of that role as I help our new member, Cora, learn to preform her tasks in the lab. As she starts her first days of work, I can definitely empathize with her position as the green new member!
Another bit of newness to me is that I made my first lab presentation last Wednesday. Every other Wednesday, our lab members gather for a meeting in which we discuss general business and then hear a presentation on the progress of one member's work, generally accompanied by a PowerPoint and heavy debate. I was a little nervous to present my work in front of the lab for the first time (how bad were they going to grill me?!) but the presentation went well, and it was a great opportunity to reflect on my trajectory for the rest of the summer.
Last Wednesday, another exciting occurrence befell the lab as film crews rolled in to take some footage of the lab at work. The paper (see above link) recently published from our lab in EMBO has gotten some local attention, so a news cast came in to interview Dr. Chai and film the rest of the lab at work. Drs. Chai, Dai, Huang, and Shilpa put a tremendous amount of work into said paper, so it is very exciting to see them getting positive feedback on their work!
As I write this, I am currently waiting for a ligation reaction to run (as a part of a molecular cloning experiment), and my timer is about to go off to send me back to the lab. More news later!
First, a brief introduction to the other members of my lab. Our lab has operated with five members for most of the summer, but we have a new lab tech who has just joined our group this week. The Primary Investigator in my lab is Dr. Chai, who is a faculty member of the WWAMI program and Washington State University's School of Molecular Biosciences. In our lab are also two post-docs, Dr. Dai and Dr. Huang, who were recently listed as the first authors on this paper (a very interesting one in the telomere field!) The other full-time member of the lab is Shilpa S., who is a Ph.D. student originally from Bangalore, India. All of the lab members have been a great help to me as I have learned to culture tissue, run different reactions, and navigate my way around the lab--I have been very fortunate that all the members of my lab are outstanding teachers. Now, I am playing a bit of that role as I help our new member, Cora, learn to preform her tasks in the lab. As she starts her first days of work, I can definitely empathize with her position as the green new member!
Another bit of newness to me is that I made my first lab presentation last Wednesday. Every other Wednesday, our lab members gather for a meeting in which we discuss general business and then hear a presentation on the progress of one member's work, generally accompanied by a PowerPoint and heavy debate. I was a little nervous to present my work in front of the lab for the first time (how bad were they going to grill me?!) but the presentation went well, and it was a great opportunity to reflect on my trajectory for the rest of the summer.
Last Wednesday, another exciting occurrence befell the lab as film crews rolled in to take some footage of the lab at work. The paper (see above link) recently published from our lab in EMBO has gotten some local attention, so a news cast came in to interview Dr. Chai and film the rest of the lab at work. Drs. Chai, Dai, Huang, and Shilpa put a tremendous amount of work into said paper, so it is very exciting to see them getting positive feedback on their work!
As I write this, I am currently waiting for a ligation reaction to run (as a part of a molecular cloning experiment), and my timer is about to go off to send me back to the lab. More news later!
Telomere Tales: Stories from a Summer of Biological Research
19 July 2010: A Lengthy Update!
This being only my second blog post, I realize I have a long way to catch you up on my research! So, here is a summary of what has been done so far this summer.
As a refresher, the goal of my research is to study the mutations that commonly occur in a gene called Stn1, which binds to telomeres and is thought to effect their length. We would like to know the effect common Stn1 mutations have on telomere length, and we propose to do this both by studying cells which already have the mutation, and by introducing the mutation into non-mutant cells and observing the result.
Orignially, we were interested in three mutations commonly found in the Stn1 gene. We sequenced many normal and cancerous cells, and found a relatively high incidence of the first mutation, a lower incidence of the second mutation (which is always found with the first mutation), and no incidence of the the third (which we had suspected, as unlike the other two, it is a very uncommon mutation and has no documented effect on telomere length). From this, we decided to focus on the first mutation, although we will continue to study the second.
Since deciding on this frame of focus, I have sequenced all the DNA I can find by designing primers to amplify the region of the gene containing the mutation, doing PCR reactions with these primers, cleaning up my PCR products and then sending them to New Jersey to be sequenced. Luckily, I had leared most of the skills I need for this process in LS1a during my freshman year at Harvard, and anything else was easy to fill-in on the go.
We were intrigued to find that tumor cells (which generally have longer telomeres) have a much higher incidence of our first mutation than normal cells do. However, a process called Telomeric TRF (one of many ways, all of which are relatively new, to measure telomere length) revealed that among tumor cells, those with the mutation did not have significantly longer telomeres than cells without, which means we did not find a direct correlation between the mutation and telomere length through this method of observation.
However, interestingly, we found that normal cells with the mutation did have longer telomeres than normal cells without the mutation. This is a point of encouragement that perhaps our hypothesis of correlation still may be correct!
At the same time, then, I have been culturing cells in which I can introduce these mutations myself in order to observe their effects while controlling for other differences (in theory, this time, the cells should be identical but for my target mutations). The steps for introducing a mutation into cells are thus:
1.)Make the mutant DNA by designing oligos that will replicate a DNA plasmid (circular DNA that contains the wildtype of my gene of interest) and introduce a mutation. Online software programs help a lot with this design!
2.) Put this mutant DNA plasmid into bacteria cells that literally have holes poked in them by a CaCl2 solution (these are called competent cells). Grow this bacteria, and let it isolate the plasmid into its genetic material. Then, collect all the DNA from the bacteria and make sure you have your target plasmid in this DNA.
3.)Apply this DNA to cells called Phoenix A cells that will uptake it and package it into viruses.
4.) Collect the viruses made by Phoenix A (they very handily dump it into the media in which they live) and infect the target cells you want to study ( I am using two cancer cell lines, neither of which orignially have mutations in Stn1).
5.) Grow the infected cells, and then check to make sure they are producing lots of your mutant protein by Western Blot.
6.) Compare telomere lengths between cells that make mutant Stn1 protein and those that have not been infected.
Today, I am doing my Western Blot of step 6 above. I have done Western before with the guidance of my friend in the lab Dr. Huang, but not alone. It seems to be working so far, but I hope it all goes correctly!
I am really excited to see if introducing the mutation will cause a difference in telomere length between cells that are otherwise identical. I will write again later to let you know what I find out!
As a refresher, the goal of my research is to study the mutations that commonly occur in a gene called Stn1, which binds to telomeres and is thought to effect their length. We would like to know the effect common Stn1 mutations have on telomere length, and we propose to do this both by studying cells which already have the mutation, and by introducing the mutation into non-mutant cells and observing the result.
Orignially, we were interested in three mutations commonly found in the Stn1 gene. We sequenced many normal and cancerous cells, and found a relatively high incidence of the first mutation, a lower incidence of the second mutation (which is always found with the first mutation), and no incidence of the the third (which we had suspected, as unlike the other two, it is a very uncommon mutation and has no documented effect on telomere length). From this, we decided to focus on the first mutation, although we will continue to study the second.
Since deciding on this frame of focus, I have sequenced all the DNA I can find by designing primers to amplify the region of the gene containing the mutation, doing PCR reactions with these primers, cleaning up my PCR products and then sending them to New Jersey to be sequenced. Luckily, I had leared most of the skills I need for this process in LS1a during my freshman year at Harvard, and anything else was easy to fill-in on the go.
We were intrigued to find that tumor cells (which generally have longer telomeres) have a much higher incidence of our first mutation than normal cells do. However, a process called Telomeric TRF (one of many ways, all of which are relatively new, to measure telomere length) revealed that among tumor cells, those with the mutation did not have significantly longer telomeres than cells without, which means we did not find a direct correlation between the mutation and telomere length through this method of observation.
However, interestingly, we found that normal cells with the mutation did have longer telomeres than normal cells without the mutation. This is a point of encouragement that perhaps our hypothesis of correlation still may be correct!
At the same time, then, I have been culturing cells in which I can introduce these mutations myself in order to observe their effects while controlling for other differences (in theory, this time, the cells should be identical but for my target mutations). The steps for introducing a mutation into cells are thus:
1.)Make the mutant DNA by designing oligos that will replicate a DNA plasmid (circular DNA that contains the wildtype of my gene of interest) and introduce a mutation. Online software programs help a lot with this design!
2.) Put this mutant DNA plasmid into bacteria cells that literally have holes poked in them by a CaCl2 solution (these are called competent cells). Grow this bacteria, and let it isolate the plasmid into its genetic material. Then, collect all the DNA from the bacteria and make sure you have your target plasmid in this DNA.
3.)Apply this DNA to cells called Phoenix A cells that will uptake it and package it into viruses.
4.) Collect the viruses made by Phoenix A (they very handily dump it into the media in which they live) and infect the target cells you want to study ( I am using two cancer cell lines, neither of which orignially have mutations in Stn1).
5.) Grow the infected cells, and then check to make sure they are producing lots of your mutant protein by Western Blot.
6.) Compare telomere lengths between cells that make mutant Stn1 protein and those that have not been infected.
Today, I am doing my Western Blot of step 6 above. I have done Western before with the guidance of my friend in the lab Dr. Huang, but not alone. It seems to be working so far, but I hope it all goes correctly!
I am really excited to see if introducing the mutation will cause a difference in telomere length between cells that are otherwise identical. I will write again later to let you know what I find out!
21 June 2010: The First Post
Hello! Welcome to the developing account of a summer spent in a molecular biology lab in Washington state. This being the first post of my summer research blog, I suppose I will begin by explaining a little bit about my project.
I was very fortunate this summer to receive a Herchel Smith Summer Undergraduate Research Fellowship at Harvard, which has allowed me to join the lab of Dr. W. Chai of Washington State University. Dr. Chai's lab studies telomeres, which are regions at the end of chromosomes that can be thought of as analogous to those plastic bits at the end of shoelaces. Telomeres are regions of repetitive DNA at the end of chromosomes that curl into special structures which prevent the cell from recognizing the ends of chromosomes as breaks in DNA. Generally, each time a cell divides, its telomeres shorten a bit until they become critically short, at which point the cell knows that it is time to stop dividing and senesces. In cancer cells (as well as in germ and stem cells), an enzyme called telomerase lengthens telomeres after each cell division so that the cell is granted some immortality. Telomerase is thus a beneficial enzyme when at work in germ and stem cells, but can be deadly when it is activated in cancer cells.
In the lab this summer, I am specifically studying one part of a tri-protein complex (called the CST complex) that binds to telomeres and is thought to control the length of the G-overhang, which is the part at the end of the telomere where one DNA strand (the strand that contains mostly G bases) is longer than the other. This G-overhang structure is critical to telomere function, as it tucks back into the double-stranded region of the telomere and forms a "knot" that protects the end of the chromosome.
The protein I am working with is termed the Stn1 protein, and its function as-of-yet is largely unknown. However, a study of the protein by Levy et al. in 2009 reported that two mutation sites in this gene are associated with longer-than-normal telomeres. Interestingly, both of these mutations create new phosphorylation sites in the protein product. A search of SNP databases online revealed that a third mutation commonly occurs in the coding region of this gene, so I am currently working with three possible mutation sites.
My major project this summer is to insert these Stn1 mutations into cells and then analyze the effect of the mutation on telomere length. As I am beginning to see, though, this project is only the jumping-off point for many other inquiries that will beg to be explored along the way!
I was very fortunate this summer to receive a Herchel Smith Summer Undergraduate Research Fellowship at Harvard, which has allowed me to join the lab of Dr. W. Chai of Washington State University. Dr. Chai's lab studies telomeres, which are regions at the end of chromosomes that can be thought of as analogous to those plastic bits at the end of shoelaces. Telomeres are regions of repetitive DNA at the end of chromosomes that curl into special structures which prevent the cell from recognizing the ends of chromosomes as breaks in DNA. Generally, each time a cell divides, its telomeres shorten a bit until they become critically short, at which point the cell knows that it is time to stop dividing and senesces. In cancer cells (as well as in germ and stem cells), an enzyme called telomerase lengthens telomeres after each cell division so that the cell is granted some immortality. Telomerase is thus a beneficial enzyme when at work in germ and stem cells, but can be deadly when it is activated in cancer cells.
In the lab this summer, I am specifically studying one part of a tri-protein complex (called the CST complex) that binds to telomeres and is thought to control the length of the G-overhang, which is the part at the end of the telomere where one DNA strand (the strand that contains mostly G bases) is longer than the other. This G-overhang structure is critical to telomere function, as it tucks back into the double-stranded region of the telomere and forms a "knot" that protects the end of the chromosome.
The protein I am working with is termed the Stn1 protein, and its function as-of-yet is largely unknown. However, a study of the protein by Levy et al. in 2009 reported that two mutation sites in this gene are associated with longer-than-normal telomeres. Interestingly, both of these mutations create new phosphorylation sites in the protein product. A search of SNP databases online revealed that a third mutation commonly occurs in the coding region of this gene, so I am currently working with three possible mutation sites.
My major project this summer is to insert these Stn1 mutations into cells and then analyze the effect of the mutation on telomere length. As I am beginning to see, though, this project is only the jumping-off point for many other inquiries that will beg to be explored along the way!
About The Author
My name is Annie Morgan, and I have just completed my freshman year at Harvard, where I plan to concentrate in Chemistry. I'm a native of Spokane, Washington and a lifelong fan of Gonzaga basketball. I love outdoor adventures--mountain biking, waterskiing, snowboarding and rock climbing, among others--and spend a lot of time at Priest Lake, Idaho. I also run competitively with the Greater Boston Track Club and attend Christ the King Anglican Church in Spokane. Cancer biology is a special interest of mine, and I hope to one day b
