Weeks 10 and 11: Department of Biochemistry, University of Iowa

August 14th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

The final weeks of my internship consisted mostly of poster work and saying goodbye to all of the wonderful members of the Baker lab.  Although I did help inject one day and helped with cloning of constructs H36 and H37, I benefitted the most from my work on the poster and review paper which both helped me understand the scope of all of the things I accomplished in these short 11 weeks.  Just listing out the majority of the skills I learned this summer was overwhelming.

I utilized several methods in order to collect data including in no particular order: PCR, colony PCR, Overlap Extension PCR splicing, Midi kit DNA isolation, Mini kit DNA isolation, gel electrophoresis isolation, Lasergene software utilization for primer and gene construct design, plasmid ligation, bacteria cell transformation, bacterial amplification, Restriction Enzyme Mediated Integration reactions, frog handling, frog hormone and anesthetic injection, frog dissections, dissection microscopy, inverted microscopy, confocal microscopy, glass microinjection needle pulling, needle back-loading, transgenic microinjection of X. laevis oocytes, embryo and tadpole sorting, gfp screening of tadpoles, tadpole processing for sectioning, tissue sectioning, slide preparation and staining, Immunohistochemistry, nanodrop DNA concentration checking, and DNA sequencing.

After discussing it with Professor Christie-Pope and Dr. Baker, we have agreed that I will attempt to return for blocks 2 and 3 of this coming school year to continue my research in pursuit of an honors thesis.  The honors thesis program at Cornell is very similar to the graduate student programs offered at the University of Iowa and other graduate level programs across the country.  In my pursuit of this thesis, I hope to prepare myself for a career in science that would not be available to me without this experience.

Summer Undergraduate Research Symposium Presentation

Summer Undergraduate Research Symposium Presentation

The last piece of my summer experience included two poster presentation sessions in week 11.  The first one included all of the undergraduates who had performed a summer research project.  There were about 160 people from a number of fields including psychology, virology, biochemistry, organic chemistry, physics, art, engineering, sociology, and more.  I got to talk to a few people who were interested in frogs and even met with a member of the admissions board for the Biochemistry department’s graduate program who listened to my presentation.  I was also able to present my poster to a few more people at the FUTURE symposium and met some of the FUTURE alumni.  It was a great experience telling people about what I learned.

I cannot express the gratitude I have for the members of the Baker Lab, the Fellows program, the Dimensions program, and all of those who financially supported this summer experience that allowed me to immerse myself in the culture of primary scientific research.  This has pushed me towards pursuing a career in research because I can see myself working in a laboratory with people who have that ingrained passion for learning which would bore most people.

Week 9: Department of Biochemistry, University of Iowa

July 23rd, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

Things are continuing to come to a close for my experience here.  I’m still working on cloning constructs H36 and H37, and the HCN1 N terminus construct H35 is ready for injection, but the amount of experiment work I do is dwindling.  This week we injected construct H27, one of Yuan’s constructs that had low expression and is one of the most important constructs for her project.  The usual tadpole sorting and routine lab activities have taken place similar to work done in the previous weeks.

I’m looking forward to injecting constructs H35 and H33 in week 10 even though I won’t be able to see the results as soon as they are completed.  I’m hoping that I will be able to take a block off during the year and work in the Baker lab to help finish up some of the smaller details of my project and put together a complete honors thesis.  However, if the H36 and H37 constructs do not show inner segment targeting as we expect one of the two of them to contain, Yuan will need to look at many more truncations of HCN1 CT which may take another summer long period of exploration in which I may or may not be able to participate.

The lab is waiting for my presentation in week 10 which I have completed and will continue to polish until Friday of week 10.  I still have to work out what snacks I’m going to bring to show how much I appreciate them taking the time out of their busy schedules to give me feedback on my project and presentation so that I’m fully prepared for the poster sessions in week 11.

I’ve also communicated more with Professor Barbara Christie-Pope and will get to see the Cornell Lab and their work on a drug that causes Zebra fish to lose neural function and lose their melanocytes.  I’ll also get to talk to a little bit about this honors thesis I’ve been mentioning so much.  She sent me the thesis that Megan Dibbern wrote in 2012 which gave me an idea as to the scope of what a thesis requires.  To say the least, it’s intimidating, but I’m learning quite a bit here and think that with a little more work in the lab and the continued support of the ever-awesome members of the Baker lab and my mentors at Cornell who encourage my scientific and intellectual growth, I’ll be able to tell a compelling story about protein trafficking in frog photoreceptors.

 

Week 8: Department of Biochemistry, University of Iowa

July 16th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

With Thursday and Friday included this week, I was able to accomplish a much greater amount of work.  Joe and I injected construct H28 for a second time because all of the tadpoles from the first injection lost their expression by the time we sectioned them and IHC didn’t reveal any GFP.  For the purposes of creating a story from this project, this isn’t a very big setback.  I also managed to finally linearize construct H33 with Not I so we’ll be injecting that in week 9.  My first attempt at ligating my insert for H35 didn’t work.  However, I have created a new H35 insert fragment which will hopefully work in my next ligation.

The needle puller used to -and I know this is a stretch (get it?)- pull needles for transgenics.

The needle puller used to -and I know this is a stretch (get it?)- pull needles for transgenics.

Joe also showed me the machine (pictured above) they use to pull the glass needles we use for microinjection of constructs into Xenopus oocytes.  The needles need to be a certain length which means we can’t just use a Bunsen burner to pull the needles by hand, like our instructors in organic chemistry lab at Cornell taught us to.  It takes much longer to position everything correctly, but the needles we pull are identical and are more likely to work in the injection process than if we used another method to make these needles.  Joe also showed me how the Restricition Enzyme Mediated Integration (REMI) reaction worked and may let me prepare H33 in next week’s injection.  In REMI reactions, the biggest potential for failure (which you won’t be aware of until about a week later after you screen for your construct) involves the very delicate and volatile sperm nuclei we work with.  If you’re not careful, the nuclei will be sheared in the process of pipetting and your tadpoles will not be fertilized in the first place, or they will not express your construct (both of which will cause more frustration than any person should experience).

Dr. Baker called a data blitz lab meeting where everyone (but myself) presented what they were working on, what their results were, and what they were excited to start working on.  This was incredibly beneficial for me because everyone spelled out very clearly what they were working on at a level that I could understand.  The reason I did not present was due to the fact that I will be presenting slides to the lab on Friday of week 10.  This presentation will be a practice run and a time to edit my poster which I will present at the Summer Undergraduate Research Conference and potentially the FUTURE in Biomedicine Research Symposium if there’s enough room for me.  My work will start to transition from performing experiments to working on these presentations and organizing my lab notebooks for Yuan after I leave.  This is a little sad as I have been having so much fun this summer working under the guidance of Dr. Baker, Yuan, Joe, and all of the other members of the Baker Lab, but know that I’ll keep in touch with them and that my work will help them form some ideas for their publication.

I also had time to meet with our very own Professor Barbara Christie-Pope and other Cornell students and alums to talk about what we were studying this summer and catch up on how everyone was doing.  Because Barbara, my advisor, is doing research in the very same building I’m doing research in, I’m able to talk to her about what I’m doing, and get a detailed explanation of what Brianna and Barbara are looking at.   I’ve made arrangements to visit the Cornell lab in week 10 and discuss with Barbara the possibility of exploring an honors thesis based on my work here this summer in conjunction with an additional block of study during the school year.

Week 7: Department of Biochemistry, University of Iowa

July 10th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

With the Fourth of July on Thursday of this week, we only had 3 work days followed by a 4 day weekend.  This was great because the Iowa City Jazz Festival was happening downtown where I got to see numerous musicians play some great jazz.  During the work week, however, I did prepare H28, H31 and H32 for confocal microscopy in Week 8 as well as preparing H28 and H33 for transgenic injection on Tuesday of week 8.  H28 needs to be re-injected because all of the tadpoles from the first injection lost their expression before we sectioned them.

I also started working on cloning the N-Terminus construct by creating fragments using primers Yuan ordered for me as well as the templates used to create each fragment with the vector being used for one fragment and the cDNA Yuan cloned by Reverse Transcriptase PCR using the mRNA isolated from Xenopus tropicalis brain and retina tissue.  After failing to get the desired fragments the first time around, we used touchdown PCR to increase the specificity of the fragments created.  Touchdown PCR is essentially Phusion PCR with a special beginning sequence.  The machine first denatured the DNA as usual, but then the first annealing temperature was 5 degrees C above the normal annealing temperature.  This ensures the primers will specifically anneal to where we want them to anneal and prevents random annealing to other parts of the cDNA.  However, this temperature may be too high for the primers to anneal at all, so for the next cycle, the annealing temperature is lowered by 1 degree C.  We repeat this for a total of 10 cycles with each cycle 1 degree C lower on the annealing step than the previous cycle.  Following this process, we then run the remaining part of the reactions as we would run a Phusion PCR.  Using this technique, I saw results on the gel that look like what I was hoping for and Yuan got very excited that we wouldn’t have to spend a week trying to get that to work.

The more I learn about how research is conducted at every level here at the University of Iowa, the more it becomes apparent that one of the most attractive qualities of studying at this institution is the collaborative spirit that permeates the majority of the programs.  The medical school trains PA’s alongside MD’s to prepare the PA’s to work with MD’s and vice versa.  There’s also the MD/PhD program which has its students working the MD program as well as in laboratories with the grad students.  The grad students work in their labs, but if they find something interesting and want to use the equipment or consult individuals of another lab or department, they’re encouraged to do so.  This connection between all of the research faculty and education granted to the students of this environment is really pleasing to my ideals.  People working hard and helping each other to progress scientific knowledge should be as easy as possible, and I think the U of I has a system in place that really lends itself to doing just that.

Week 6: Department of Biochemistry, University of Iowa

July 2nd, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

2013-05-31 11.08.50

Fixing tadpole tissue in the fume hood.

Week six held many good things.  We finally have data. Hooray!  Yuan took some of the slides I had prepared with the tadpole sections to the confocal microscope where we took pictures of construct H30 expression.  It was so nice to see the results up close and with the greater resolution of the confocal.  What made the experience even better was seeing some of our tadpoles that didn’t glow bright green under the inverted microscope show GFP expression when we stained the slides using Immunohistochemistry (IHC).  Dr. Baker said that her attempts to stain for GFP which was not fluorescing brightly did not result in specific staining of GFP which made our results even more exciting.

Sectioning tadpoles in the cryostat.

Sectioning tadpoles in the cryostat.

Beyond receiving these exciting results, I also managed to attend a panel with representatives of the MD, PA, and PT departments of the University of Iowa where they talked to us about the unique aspects of each program distinguishing them from other MD, PA, and PT programs from across the nation.  But another really great networking opportunity came from casual conversation in the Baker lab which resides in the 700 core containing the Washington and Weeks labs.  Sarah was asking me what I planned on doing and she talked about U of I’s MD/PhD program which is called the Medical Scientist Training Program (MSTP).  Sarah asked a member of the Washington lab who was sitting near us about the program and he responded to her questions.  After Sarah had left, a member of the Weeks lab came over and said that he and another member of the 700 core were also MSTP students and that I could approach any one of them with any questions regarding the MSTP and they could get me in to talk to the director of the MSTP.  Although only 10 spots are open each year, the MSTP program sounds very interesting and challenging.

Sarah (left) and Yuan (right) hanging out at the end of the day.

Sarah (left) and Yuan (right) hanging out at the end of the day.

Dr. Baker called the first lab meeting where Modestos presented his work on finding proteins other than TRIP8b that complex with HCN1.  He was using a process called Immunoprecipitation (IP).  The problem he was having was finding large bands at 25 and 50 kDa in his silver stained SDS PAGE results.  He thought these bands were the light and heavy chains of the IgG he used to bind HCN1 and tried different procedures to try and remove the IgG in numerous ways.  We sat in the meeting scratching our heads trying to figure out why it wasn’t working.  Then after the meeting, they looked at the Western Blot results and realized the IgG that was supposed to only bind HCN1 also bound 2 other proteins and the IgG wasn’t specific.  This meant that the two bands that were found in all of Modestos’ experiments were the two proteins that were being bound by the IgG and Modestos needs to figure out a solution to this problem.

 

Week 5: Department of Biochemistry, University of Iowa

June 24th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

This week was markedly better than last week.  I got my ligation of H33 and H34 to grow colonies and found in the isolation of the plasmids that constructs H33-11 and H34-3 looked clean and ready for linearization.  These constructs were submitted for sequencing on Thursday and may be injected on Tuesday of week six if the sequencing shows no point mutations in my constructs and Sarah doesn’t need to inject three of her six prepared constructs.    We’ll see what happens on Monday.

I also prepared some of the bigger tadpoles from constructs H30 and H28 for sectioning on Monday.   This process involves many steps; the first being putting the tadpoles into a formaldehyde solution to prevent the proteins and cells from moving or decaying before we look at them.  After removing the tadpoles from the solution, we then prepare them for freezing by shaking them in a sucrose solution.  The sucrose will prevent water molecules from crystalizing in the cells which would cause the cell to burst and then we wouldn’t know what was part of one cell or another.  We then change the solution to a mixture of the sucrose solution and an Optimal Cutting Temperature (O.C.T.) compound which allows the tissues to be frozen and then washed away after we use a Cryostat to cut sections of the tadpole roughly 7 micrometers thick.  We’ll look at these sections and find our retinas which hopefully contain photoreceptors expressing our protein.

Preparing tadpoles for sectioning.

Preparing tadpoles for sectioning.

However, before we froze our tadpoles, we examined the eyes to check if the photoreceptors lost expression of the constructs.  Some of them looked completely dark instead of the bright green florescence we were hoping for.  Yuan suggested that after we section the dark eyed tadpoles, we should stain for GFP so we’ll be able to see if the GFP lost its florescence or if our construct simply lost expression in the eye. It sounds like that will be left for Thursday or Friday of next week.

Some exciting news (for me anyway) comes in the form of a new assignment.  I’ll be looking N terminus of HCN1 of Xenopus tropicalis to see if there is a targeting signal contained within that section of HCN1.  From my reading, other proteins do not seem to contain messaging signaling within this region, but because the signal recognition pathway is poorly understood, it seems very likely that the N terminus could very well contain some form of signaling that we haven’t observed yet.  However, because Yuan hasn’t worked on the N terminus of HCN1, we don’t have a template plasmid to work with.  This means that she needs to isolate the mRNA of X. tropicalis from brain and retina tissue to perform a Reverse Transcriptase-PCR to get the cDNA for all of the proteins expressed in these tissues.  Once we have the cDNA, we’ll use touchdown and/or nested-PCR to attain the N terminus fragment we want to introduce to our vector.  Touchdown-PCR involves starting at the highest temperature that will allow our selective primers to anneal to the cDNA and cycle at progressively lower temperatures to amplify the N terminus fragment where the nested-PCR involves two reactions where one set of primers are used in the first reaction which will produce a fragment we know will contain a little more than our desired N terminus, but then follow that reaction with primers which should contain exactly our N terminus fragment.  I’m excited to see if this works.

Another huge boost to my week came from Dr. Baker and Yuan when they told me that I will be included as a middle author on the paper they are planning on publishing involving HCN1 trafficking in the photoreceptor.  Not only does this look good on a resume for anything related to science, but it supports my idea that I’m aiding in the process Yuan has been working on for 3 years.  I’m learning quite a bit and look forward to the second half of this experience.

Week 4: Department of Biochemistry, University of Iowa

June 19th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

This week was a mixed bag of good and bad as far as the science goes. We injected 3 more constructs (H29, H31, and H32) which was awesome with a reasonable and functional number of tadpoles expressing our constructs.  Our earlier construct tadpoles are now huge and will be ready for analysis in week six assuming the expression of the constructs doesn’t disappear.  This has happened in the past for Yuan, but I’m hopeful it’ll be alright.

The not so great news was our H33 sequencing results showed what we had was not our construct, but rather a random strand of DNA that somehow managed to come out of our ligation.  This may have been due to a low concentration of H33-fragment 1, but we’re not entirely sure.  We’re working through a couple troubleshooting steps including running gels to check the size and purity of the fragment.  We’re also digesting the fragment in high fidelity NEB enzymes instead of mixing high fidelity with regular enzymes.  We want to digest the fragment with the same enzymes the vector is digested with so we can insert the fragment into the vector.  All NEB enzymes work in certain conditions like whether BSA is required, and what temperature will activate the enzyme.  These enzymes also have different functionality in one of four buffers.  We were using NotI-High Fidelity(HF) and AgeI in NEB buffer 4.  NotI-HF has 100% functionality in buffer 4, but  AgeI has only a 75% functionality rating in buffer 4.  On the other hand, AgeI-HF has a 100% functionality rating in buffer 4.  So we’re trying the digestion with NotI-HF and AgeI-HF in buffer 4 this time around.

My construct, H34 is also having some troubles.   I started the cloning process on Sunday and by Friday had determined that I needed to start over because I was convinced by that point that the H34 insert that I had created was not what I wanted it to be.  So starting in week 5, I will start the process again with Phusion PCR of my predicted H34 fragment.  Let’s Hope for the best.

Sarah was on vacation and Dr. Baker was at a conference this week and their absence was noticed.  It really didn’t have anything to do with the less-than-progressive state of affairs in my research, but the atmosphere of the lab’s social environment was a little less robust than usual.  Joe, Madestos, Yuan and I made up for this by going on a lab lunch trip on Thursday where I was introduced to the Pie Shake at the Hamburger Inn.

Hamburger Inn's menu featuring the Pie Shake.

Hamburger Inn’s menu featuring the Pie Shake.

I would have taken a picture of the actual product, but I was just too excited and forgot about it completely.  They take a slice of pie (I chose a raspberry, apple, and rhubarb pie) and blend it into a milkshake.  Now I’m not one to miss a meal, but I didn’t eat dinner that night because I was still full from the lunch.  My ability to perform tasks in a normal state of mind after consuming the entire thing as well as a lamb pita sandwich from Oasis was… inhibited, so of course Joe and Madestos gave me a hard time about it.  All in good fun, of course.  But it didn’t stop there.  “Dave! Let’s go get a Pie Shake,” or “ You look like you could use a Pie Shake” have been thrown around more than a few times since the incident and I don’t think I will go anywhere by the end of my time here.  But it’s all in good spirits and reminds me of my own family dynamic, so it’s tolerable at the very worst.

The time I have spent here has really made me seriously consider a career in research.  It is a pleasure to work with the member of Dr. Baker’s lab, and it’s great to be able to listen to their conversations and be able to contribute even a little input that may be helpful.  My ability to actively participate in these conversations and contribute to the lab is entirely dependent upon my experience at Cornell.  My desire to perform research in a laboratory will now fuel my work ethic at Cornell in a way that I did not have before this experience.  The future seems real.  I can actually imagine what working in a lab will be like because –and I know this is a stretch, but bear with me- I have had a little experience working in a lab. It’s hard to believe, I know, but it’s honestly invaluable experience that fills the gap that’s inevitably left in undergraduate classes.  However, class is not devoid of value and gave/gives me the background and theory required to participate in research.  It’s just the act of being in the environment where you participate in primary research gives a unique perspective I’ve only achieved through immersing myself in the environment.

Week 3: Department of Biochemistry, University of Iowa

June 10th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

View from the overpass on my walk to my sublet.

View from the overpass on my walk to my sublet.

This week was referred to as “transgenic week” by the lab members due to the fact that we injected sperm nuclei (which had been modified by the reaction of the nuclei with two of our linearized plasmid constructs) into eggs this week.  And when I say eggs, I mean about five hundred for each construct.  Tuesday was injection day and instead of labeling lots of tiny micro-centrifuge or PCR tubes and pipetting small amounts of liquid between these small tubes, the day was spent using tiny, glass needles back-filled with the REMI reaction mixture and a syringe pump, to poke relatively big holes into about a thousand eggs.  After that experience, we then had two sorting periods where we took the dead embryos or unfertilized eggs out of the Petri dish so the death signals from those sick cells wouldn’t kill the few that had successfully been fertilized.

Joe fertilizing frog eggs and showing me the process on the monitor.  He's really good at this.

Joe fertilizing frog eggs with the REMI reaction and showing me the process on the monitor. He’s very good at this.

I’m proud to say that my first injection period (although I took about 3 hours compared to the 3 years of experience guy’s 1 hour) I was able to get about 70% of Joes surviving embryos in the first day.  Yuan, Sarah, and even Dr. Baker were impressed that I was able to attain a number fairly comparable to the graduate student’s success rate.  The members of this lab have been unbelievably supportive and understanding and have made this experience overwhelmingly positive.

I also started working on construct H34 by creating the H34 fragment via PCR.  I came in on Sunday to sort tadpoles and begin the digestion of H34-fragment 1 (H34-f1) in preparation for ligation on Monday. Yes, you read that right.  Tadpoles.

Tadpoles.

Tadpoles.

After injecting the REMI reaction on Tuesday, not even a week later they already resemble tadpoles and swim around when you stimulate them with a pipette.  It blows my mind that they develop this quickly and that I got to see it happen from the moment we fertilized them.  Now we’ll get to see next week if the gene we inserted is being expressed in at least 10 of them.  The transgenic process practiced in this lab isn’t very efficient, but it’s fast and relatively cheap so we just inject about 500 eggs and hope that 10 of them express our gene.

The FUTURE program offered some meetings this week for the undergraduates.  One meeting went over the graduate school programs offered by the University of Iowa which gave information on who to talk to at what point, what to include on the applications and when to ask who about how to apply, and the various advantages the U of I graduate programs had over other graduate programs.  The presentation was given by Dr. Spitz, a member of several admissions boards and oxidative stress in cancer biology and toxicology researcher who was very vibrant and able to show his excitement for the programs and his research.  There was also an informal reception allowing the undergrads to mingle and talk to one another where I met Brianna from our very own Cornell for the first time as well as some other students from Luther.  We had a very good time talking about the projects we are working on this summer and also having a few laughs about non-research-related things as well.

This next week we’ll be injecting two more constructs while I continue to work on H34.  There is a lot of work to do, but it’ll be no problem with the continued support and guidance from the lab members.

Week 2: Department of Biochemistry, University of Iowa

June 3rd, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

BSB reminds me of M.C. Escher's Relativity painting.

The BSB reminds me of M.C. Escher’s painting Relativity.

This week was noticeably more rhythmic than last week.  The protocols I performed I had already done the week before and was much more comfortable than week one where absolutely everything was new.  The molecular cloning process we’re performing is done by putting different parts of the C-terminus of HCN1 onto ACTx reporter protein sequence.  By inserting this construct DNA into frog sperm and fertilizing a frog egg, we grow tadpoles that express the construct.  The photoreceptors of these frogs are then examined on a cryostat which allows us to follow where the construct protein is being trafficked and indicates whether or not the selected section of the C-terminus of HCN1 contains a trafficking signal which directs the protein away from the outer segment.

It's funny because the cart didn't actually smell.

It’s funny because the cart didn’t actually smell.

Yuan has had me working on two different sets of DNA sequences containing different combinations of HCN1.  Yuan walked me through the steps to create constructs H31, H32, and H33 from the beginning while at the same time, Yuan had already started, and had me continue, to work on constructs H28, H29, and H30.

The idea Yuan’s and I are implementing goes something like this.

Given a template plasmid already constructed (H23 for H28, H29, and H30 and H27 for H31, H32, and H33) we select which parts of the template we amplify in Phusion PCR by selecting primers which give us the strands we desire.  After the PCR, the construct fragments, or pieces to be inserted into the final plasmid, are evaluated by agarose gel electrophoresis to make sure the majority of the fragments are the correct length and then isolated from the gel by a Thermo isolation kit.  We then send off some of the fragment for DNA sequencing analysis to make sure there aren’t any point mutations in the majority of the sample.

The fragments then get digested in restriction enzymes which clip the ends of the DNA in preparation for ligation with the vector.  The fragment and vector are then added to TOP10 cells in a process called transformation and the cells are cultured on agar dishes overnight.   We then check the size of the plasmids to make sure no weird fragment lengths were inserted into a vector.  Once the size has been confirmed agarose gel the DNA is isolated with the Thermo kit.  To make sure no point mutations occurred in the transformation stage, we send the plasmids off for another DNA sequencing.  Once we know there aren’t any point mutations in the plasmid, we digest the plasmid in restriction enzymes to linearize the DNA in preparation for transgenic insertion into the sperm DNA of Xenopus laevis or African Clawed Frog.  We check the linearized DNA by running it through a gel against the supercoiled version of the DNA to make sure the digestion was complete.  If any of the DNA isn’t digested, it will travel further in the gel and can be omitted from the DNA isolation process.

Working on a Midi kit DNA isolation for H31, H32, and H33

Working on a Midi kit DNA isolation for H31, H32, and H33

Checking the Quick Tip Column from the Midi kit DNA isolation

Checking the Quick Tip Column from the Midi kit DNA isolation

In week 3, we start the transgenic process on Tuesday by inject the H28 and H30 constructs into thousands of eggs.  Then there are several sorting periods over next few days of the week which ensures at least some of the frogs develop correctly.  Apparently, if sick or dead fertilized eggs are left in with the healthy ones, all of the eggs die.  The process is very inefficient and the vast majority of the frogs will die with one period even called the “death night” where the majority of the organisms will die off.  Although this makes me a little sad and scared, at least they gave me some fair warning that this is going to happen and that it may not be entirely my fault if a lot of them die.

Some exciting news came when Yuan told me that I will be responsible for the entire creation of construct H34! I’m responsible for scheduling my time appropriately and preparing the construct for transgenic insertion on June 25th.  This shouldn’t be too difficult as I’ve already been through the process about one and half times by now, but it’s really exciting that I’ve reached a point in my education where people trust me to perform primary research and will rely upon the results I collect to form conclusions and may even include it in their publications.

The fellowship I’m participating in is running virtually parallel to the University of Iowa FUTURE in Biomedicine program which is a summer program for fellows (usually professors) of small undergraduate schools (like Cornell) can bring a student to work with a professor in their lab over the summer.  The FUTURE program has seminars and talks for the undergraduates participating in this program and I am lucky enough to be able to attend these talks.  The most recent event was the undergraduate research survival skills workshop where they told me how to keep a lab book and how to give a powerpoint  and poster presentation.  BIO-141 with Professor Barbara Christie-Pope (who is a “Senior FUTURE Fellow” –the “Senior” part she especially adores- in Robert Cornell’s Lab) and BIO-205 with Professor Craig Tepper gave me all of the information covered in the seminar reassuring me that my time at Cornell is well spent and preparing me for a career in Bioscience if I choose to pursue it.

An amusing anecdote about the lab would have to be what I’m calling the -80°C Freezer Re-Organization Debacle.  Madestos (a.k.a. “The Angry Greek”) is the lab manager who knows where everything is and how most of the stuff works and had reorganized the -80°C freezer and thought everyone knew where things were.  While I was running a Thermo DNA isolation kit, a member of another lab came over to Modestos and said, “I think Sarah was calling for you” as Sarah was in the -80°C looking for something.

Joe walks over to see what was wrong because Modestos was busy with some protein work.  After a few minutes, Sarah comes back down the hall to our lab looking a little… irritated followed by a giggling Joe.  I started laughing as Modestos asked Sarah with his Greek accent, “Is everything okay?  Were you calling for me?”

She responded sharply, “No.  I was cursing you for putting my stuff where I couldn’t find it.” And before Modestos could respond and without really breathing, she added, “I was so freaked out.  You can’t do that to me.”

After that there was the expected basic argument concerning what organization system had been agreed upon.  This lasted for much longer than was necessary for clarity, but just the right length to be amusing to no end.

There’s also been an unreasonable amount of rain recently and the rivers are running pretty high.  Joe told me the University of Iowa website has a link with information concerning the high rivers.

http://now.uiowa.edu/keywords/flood-updates-2013

The river's running pretty high.

The river’s running pretty high.

Week 1: Department of Biochemistry, University of Iowa

May 28th, 2013

David Yamaguchi ’15, Dimensions Fellow in Research

Wow. The first week here in the Bowen Science Building (BSB) of the University of Iowa has been a great experience. The people involved, the location, and the material I’m learning about and working on have all exceeded my expectations. I had met with some of the people in the lab before the internship started and got a brief overview of what we were going to be working on this summer.  It’s all about the photoreceptor cell in Dr. Sheila Baker’s lab.  The photoreceptor cells are compartmentalized into 4 distinct areas with the modified cilia outer segment, signal processing and cell metabolic processing inner segment, the DNA containing nucleus, and the synaptic region responsible for sending neurotransmitters to other neural cells.

A diagram of the photoreceptor cell. Credit- Baker, SA http://www.biochem.uiowa.edu/baker/index.html

A diagram of the photoreceptor cell.
Credit- Baker, SA
http://www.biochem. uiowa.edu/baker/index.html

Dr. Baker is interested in how different proteins are transported selectively into their distinct regions of the photoreceptor cell.  According to the research she has conducted, proteins are automatically directed to the outer segment.  For direction to any other section of the photoreceptor cell, the protein requires a signalling sequence.  I’m working with Yuan Pan, a graduate student, on her project to figure out what part of the C terminus of the HCN1 protein directs the protein to the inner segment of the photoreceptor cell.

Before I learned all of that however, I first walked into the lab where Joe, a friendly research assistant who plays a lot of video games outside of the lab, greeted me and then helped me get registered and trained on how to use various equipment and the safety protocols of the lab.  He then introduced me to the rest of the lab. Sarah, a graduate student working on another project, is very bubbly and talks at rates my brain has trouble processing.  Vasily is a post doc working on another project.  And Modestos is the originally Greek research assistant who is older and gives me advice that I don’t think I will follow.  For example, I was told that when culturing bacteria, I should add in some ethanol to make the colonies happier so they will grow bigger.  If you know anything about culturing bacteria, this advice would seem very suspect to you and would realize that he was kidding.  He has a lot of fun and everyone seems to know him at the coffee shop in the hospital (which is connected by sky walk to the BSB).

These people work together very well and sometimes talk in language that my inexperienced mind interprets as nonsense.  However, after spending my first few days reading and training on the safety protocols and various equipment, I’m starting to understand more of what’s going on.  I started running kits, culturing bacteria, and running gels all relating to the plasmid preparations that are universal laboratory skills in biosciences.  A new experience for me was designing primers on the computer and then getting them two days later.  Better yet, I found that the primers I designed with Yuan’s guidance, actually worked in Phusion PCR amplification of segments of DNA we’ll use to inject into frog embryo’s in the second or third week.

Professor Tepper’s Cell and Molec. class really gave me a solid foundation for biochemical lab work which prepared me for lab procedures to a greater degree than most of the lab members were anticipating.  The major difference I am finding between class and this internship would be the time limits.  Because there isn’t an 18 day limit to this lab process, I can take more time to fully understand what I’m doing and have a much more relaxed lab experience.  This will more than likely change as I am given more and more responsibilities, but this introductory period is very nice.

Another one of the things I wanted to gain from this experience was whether or not I was interested in pursuing a career in research, medicine, or some combination thereof.  I have found that the social atmosphere of this lab to be unbelievably comfortable and have no trouble seeing myself working with people like those who populate the Baker lab.  The work can be tedious, but I think it will be rewarding beyond my current comprehension.

I have to apologize for the lack of pictures, but this first week was very busy and the opportunity for pictures were few.  I’ll be sure to upload many next week to make up for this visually bland post.

 

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