Week of 5/14/17

Today was Signature Day! All of the STEM interns and Signature creators presented their projects to a crowd of students, faculty, grandparents, and special friends. It was a lot of fun to show everyone the results of my research and help them understand my project.

I presented at 9:00 in the morning to a large crowd crammed into Snell 102. After Nana and Michaela finished summing up their projects, I got up to give my presentation. Everything went smoothly, and I got a lot of great questions afterward!

This entire year has been incredible. I have loved working at RPI in the CBIS High School Scholars Program. Yi and Dr. Royer are both brilliant and kind people, and their guidance and wisdom have really enriched my senior year.

If any Emma students (current or prospective) are reading this post, I hope you strongly consider doing a STEM internship as soon as you are able to. It was so much fun to obtain so much knowledge and lab experience. It definitely made senior year much more enjoyable!

Thank you to everyone who has helped me move forward with this project. I want to give particular mention to Yi, Dr. Royer, Ms. Mossop, Ms. Biggins, Mr. Calos, the shuttle drivers and to all the people who made little contributions to my experience along the way. Thank you!

Week of 4/23/17

Today was prep for my poster session, which is this Thursday. I am very excited to present at RPI as part of the CBIS High School Scholars Program. Rather than describe my preparations, I thought I would post my notes here for you to read and give an idea of what I am going to say this Thursday when I present.

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Protein folding is one of the most important biological processes

For many proteins to perform their biological processes, they must fold into a specific 3D structure

This process is called protein folding – are interested in how folding processes throughout this process

If we want to study protein folding, we need to perturb this process – otherwise it is stuck at equilibrium

There are sever types of perturbants

Chemical denaturants (urea, guanidine hydrochloride)

Temperature, changing pH

But in our lab, we use pressure

Talk about how pressure perturbs folding – due to internal cavities in internal structure

In the folded protein, packing is imperfect

In folded structure, due to imperfect packing of atoms, there are solvent excluded cavities (where water cannot enter)

Pressure acts to eliminate these cavities (bc pressure minimizes volume) and unfold the protein

From this pressure study, we can obtain volumetric properties of the protein

Pressure only acts on the cavities – cavities are different for each protein – this way, pressure perturbs cavities of different proteins differentially

Pressure effect has local feature, therefore it reveals more detailed information regarding the protein folding process

Why not chemical? Temperature?

Chemical has global effect

Protein we study: PP32

PP32 belongs to acidic nuclear phospho protein family

Tumor suppressor – good for cancer research (biological significance)

We’re more about physical significance

Leucine-rich pp protein

Repeat protein (5 repeats – arrows) conserves leucine residues within repeats

Repeats are similar in structure and sequence (look similar – leucine rich at one part of arrow? Same 

for next arrow)

Cavity in center of structure (empty – no water) spheres

We study this protein because it is a repeat protein so its overall architecture is linear and simple – easy to study

Simple linear architecture abundant in local contact, lacking global interaction (by folding studies)

Protein folding is matter of structural energetic interaction

Also good for NMR – use fluorescence (tryptophan, terasine, phenylalanine)

Only rings can accept fluoresce

Quantum yield – how much light is emitted (give 10 photons, how may do you get back)

Tryptophan has higher quantum yields than other two – so low we can’t see it

Unfortunately, there is no tryptophan in this protein

Tryptophan residue introduced at c terminus for fluorescence measurements

Yellow c terminus

PP32 has two capping domains – on two termini (stabilize protein)

Two black sticks are two residues: aspartic acid 146 and tyrosine 131

Numbers are residue number (area around first top loop is approx. 18)

Why are they there? Side chains close to one another-  hydrogen bonding between the two

Keeps the structure stable and stabilizes the entire protein (there naturally)

How the fluorescence works

Tryptophan likes to be excited with wavelength 290 nanometers

Gives emission spectrum which are dependent on micro environment around that residue

Environment is determined by protein structure

Therefore when proteins are folded and unfolded, we get different emission spectra (different 

environment)

Folded is lower peak –

Usually, folded has higher peak, but in this case – we think there are histanine residues around the 

tryptophan which quenches the fluorescence (when it is folded) – when protein unfolds, quenching effect dissipates

Folded and unfolded states have different emission spectra – can be differentiated

With the mutations (change Y131 to F and D146 to L) – we can break up the hydrogen bond and unfold the protein

Take fluorescence measurement, increase pressure to new level, protein unfolds to some degree, let it 

reach new equilibrium, take next fluorescence measurement, repeat

That’s how the instrument works -> water pumped in and increases pressure

For each measurement, we increase the pressure and protein starts to unfold -> takes time to unfold

Excitation wavelength at 290 nm

In fact, when Yi increased pressure, he didn’t take full emission spectra – takes too long

Just one – 340 nm

Averaged equilibrium values for intensity graph

Protein dissolved in urea (helps to unfold), water, bis-tris (pH buffer) – more denaturant added, easier for pressure to unfold protein, DTT (reducing agent – prevents proteins from aggregating)

Urea facilitates protein unfolding

pH 6.8 20 degrees

What we did: took emission spectra of this protein after the system reaches equilibrium

How do you know it reached equilibrium?

Monitored intensity at 340 nm as function of time -> signal doesn’t change anymore

Take intensity at 340 nm and plotted it as a function of pressure – going past 340, intensity is the same (asymptote)

At each pressure, take the value at 30 nm -> plot 340 as function of pressure

Sigmoidal (s curve)

Unfolded – higher value

Folded – lower value

Two state model – looks at unfolded versus folded  (we follow two state mode to analyze data)

Transition state is population weighted average of two states – percent unfolded and percent folded average gives transitional value

Delta g value varies with amount of urea

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I hope my notes are somewhat intelligible. I am very excited to present my poster!

Week of 4/2/17

Today was my first day back in the lab in over a month! I was really eager to get back. Yi finished his thesis defense, which is awesome, but that also meant that e was really tired so we ended up just doing a few simple tasks in the lab today.

First, we prepared some cultures and began growing bacteria. I pipetted three milliliters of LB broth into a tube under a flame, and repeated this process five times. Then, Yi gave me six different samples of bacteria to pipette into the individual tubes. Again, we performed this process under the flame.

Once all of the bacteria had been loaded into the tubes with growth medium and labeled, we placed them in the fridge to grow. If we had wanted them to grow faster, we could have put them in an incubator shaker for two to three hours at thirty-seven degrees. When bacterial samples are shook, the agitation incorporates oxygen and evenly distributes nutrients to all the bacteria, helping promote faster growth.


An incubator shaker. https://megadepot.com/product/ika-works-3940100-ks-3000-ic-control-incubator-shaker

After leaving the bacteria in the fridge, Yi and I had no more work to do. We offered help to another graduate student working in the lab, and she asked us to prepare 250 milliliters of 3 M NaCl. It was quite simple to calculate how many grams of powdered NaCl to use, I have outlined my calculations below.

3 mol / L * (.250 L) = .75 mol NaCl wanted
.75 mol 8 (58.44 g / mol) = 43.83 grams of NaCl

I weighed 43.83 grams of NaCl and poured it into a liter flask. Then, I added enough distilled water to bring the total number of milliliters of solution to 250 mL, and then shook and swirled the flask vigorously until the solution was completely dissolved.

I was very happy to get back in the lab this week. Even the simplest tasks are really fun, and I appreciate them even more as the year winds down. Can't wait for next week!

Week of 3/26/17

Yi had his thesis defense today, so I didn't end up going to RPI. I can't wait to get back!

Week of 3/5/17

Yi had a meeting today so I couldn't get to RPI, but I eagerly anticipate our next session!

Week of 2/5/17

This week's meeting was great! Yi and I used the agar growth mediums we prepared a few weeks ago to plate the bacteria. We unwrapped the three petri dishes from their parafilm seals and set them aside. Next, we gathered the materials necessary for plating the bacteria- a sterile loop, Bunsen burner, and the bacteria culture.

Once we had all of our materials, it was time to plate the bacteria. We turned on the Bunsen burner and waved the loop quickly through the flame to ensure the surface was sterile. Making sure to keep all activity under the burner (again, to prevent contamination), we stuck the loop into the tube of bacteria and picked up a small quantity of bacteria. Then, we transferred the bacteria onto the agar plate using a criss-cross pattern, as shown below.




Bacteria on agar criss-cross pattern. http://teachersinstitute.yale.edu/curriculum/units/2010/3/10.03.01.x.html

Why were the bacteria plated in a criss-cross pattern? This is to ensure that the bacteria do not grow clumped and crowded with one another. This pattern not only ensures a more equal distribution of resources, but also makes it so that individual colonies can be observed as they grow, rather than a mass collection of colonies impossible to study.

It should also be noted that our bacteria culture included a small amount of antibiotic within its medium. The bacteria we were studying possessed resistance to the antibiotic, and thus were not affected by the drug. This again was a preventative measure against contamination- any bacteria that somehow enters the chamber will be killed by the antibiotic, ensuring that the only growing bacteria will be the desired strain of study.

By observing the growth patterns of our strain of bacteria, we can see the expansion of individual colonies. Although I have performed experiments like this before, it was great to be exposed to more lab techniques and practice scientific skills in a real laboratory setting. This meeting was great, and I cant wait for next week's internship meeting!

Week of 1/30/17

Time for the interview! After working in the lab and placing our bacteria in the growth medium that we prepared last week, Yi and I sat down for a few minutes so that I could ask him some questions. Enjoy!

Interviewer: Molly Smullen, Senior at Emma Willard (M)

Interviewee: Yi Zhang – graduate PhD candidate at RPI (Y)

M: Yi, can you provide a short summary of the work you do at CBIS?

Y: I study protein folding with NMR fluorescence and Saxs under high pressure.

M: What does studying proteins entail?

Y: To study protein folding, you need to break the balance of proteins in the unfolded versus folded states to understand what propels the protein into its folded state. Whatever parameter you use to break this balance is called a denaturant. Common denaturants are chemicals (such as urea), pressure, or temperature. In my lab, we use high pressure as our denaturant, because we believe it is a softer denaturant, and targets protein structure locally. Only the cavity, where proteins are not perfectly packed, is targeted.

M: What are some different techniques for studying proteins?

Y: You should always use a wide range of biophysical methods for studying proteins because they each reveal something different. For example, Sexs provides information about the overall conformation of the protein, and informs you of overall change in protein shape. Fluorescence is also used to study the general form of proteins. NMR provides more detailed information because its resolution is resolved to the atomic residue. Therefore, you are provided with sequence-based information.

M: How did you become interested in this sort of work?

Y: I have always been interested in biology, since high school, or even middle school. It was very natural to take on this research path, and proteins are a hot topic right now. Also, the techniques you use in protein studies are widely used, so you are trained for many different areas when you study proteins.

M: What implications does your research have in the scientific community/the world?

Y: It’s always good to study protein folding mechanisms because proteins are the major functional components of our bodies. Drug design is also becoming more target-based on protein structures, so this research could provide some insight and guidelines for future drug design.

M: What are your future plans?

Y: I am planning on going to law school and becoming a patent lawyer.

M: Why did you open your doors for an intern?

Y: I am a student, so I know what such an extracurricular activity means to a student who is eager to learn an explore that interest. It is my pleasure and honor to help introduce students to their passions. My father and grandfather were also teachers, so it is good for me to share my knowledge with others, especially younger students.

M: Is there anything you would like to add?

Y: Good luck. When you go to college, you should definitely study, but don’t only study. Do some extracurricular activities, and not just academic ones. Be social, develop interpersonal skills. College is a great time for you to explore and grow into an adult. Study, but don’t be a nerd. Grow into your own person, and have fun!

M: Thanks, Yi. I really appreciate it.

Y: You are welcome.

Great meeting this week, and Yi really provided some valuable knowledge and insight. I can't wait to get back in the lab next week!

Week of 1/23/17

Today was my first day at RPI of 2017! My internship is going swimmingly and I cannot wait to continue my work this semester.

This meeting was a preparation session. Yi and I are planning to grow some bacteria, but first had to prepare the growth medium in order for them to thrive. We mixed 50 grams of agar powder into 2 liters of deionized water and divided the dissolved mixture into two flasks. Additionally, we poured some of this solution into a smaller jar for a median solution. Finally, we added 2.5 grams of another agar powder into 60 milliliters of water.

Once our solutions were prepared with the correct ratio of powder to water, we brought all of the flasks and jars downstairs to the autoclave. An autoclave is a heated pressure chamber that is used to sterilize media to be used in industrial processes. In our case, we autoclaved the agar to sterilize and prepare it for the bacteria. I have included a diagram below that outlines how an autoclave actually works.



The mechanics of an autoclave. http://www.used-autoclave-s.com/autoclave-process/

After placing the agar in the autoclave and setting the timer for a fifteen minute cycle, Yi and I retreated to his office and waited for the temperature and pressure to rise within the chamber. Once this waiting period ended, we moved back down to the lower floor and removed our agar flasks from the chamber.

Following their extraction from the autoclave, the flasks and jars of agar needed to cool until they were comfortable enough to handle. We placed them in a bath of cold tap water and waited for about ten minutes until it was not painful to pick them up. Next, we gathered four small dishes and their lids to pour the agar into. After pouring a small sample of liquid agar into each dish and labeling each one, we wrapped the dishes with parafilm as a seal and set them upside down in the refrigerator. Placing them upside down ensures that the condensation that rests on the inside of the lid does not fall into the agar and corrupt the sample.

This internship meeting was so much fun. I cannot wait until next week, when we will place the bacteria in our growth medium!

Week of 12/5/16

This was my last internship meeting of 2016! Today was another hands-on day, so it was a lot of fun. Yi and I continued our use of the fluorescence spectrometer to determine the intensity (measured by absorbance) of folded versus unfolded proteins at various urea concentrations. To begin, I had to calculate the number of milliliters of buffer to add to the 1.1 grams of peptide.

9.7 milligrams of peptide - molecular weight = 2.9 kDa = 2900 grams per mole

9.7 * 10^-3 (1 mol / 2.9 * 10^3) = 3.34 * 10^-6 moles of protein

Final molarity should be 70 * 10^-6 M

(3.34 * 10^-6) / x = 70 * 10^-6 M

x = .048 L = 48 mL of buffer necessary

Following our calculations, we prepared our sample of protein and buffer, adding a predetermined concentrations of buffer and urea (of the sample prepared the previous week). The combined sample was pipetted into small cuvettes and placed into the large fluorescence spectrometer.

I should note that preparing the fluorescence spectrometer was no small task. Yi told me that the pressure had to be moderated very carefully, using a pump to add or remove water from the inside of the system. Traditionally, the user will turn a wheel using their hands to pump in more water, but two weeks before my visit, RPI had switched to a computer modulated pump system. Unfortunately, Yi was unfamiliar with the new computer system. We asked several other grad students in the lab for assistance, but no one could figure out why the system was malfunctioning!

Fluorescence spectrometer diagram. Image taken from: http://www.mpip-mainz.mpg.de/62430/Fluorescence_Correlation_Spectroscopy

Because of our computer troubles, we could not finish our experiment. Yi had to head to his chemistry seminar and I had to catch the shuttle back to Emma Willard. Although our experiment time was cut short, we had a lot of fun and I learned a lot about how fluorescence spectrometers work, and how delicate they are. I'm very excited for my first internship meeting of 2017!

Week of 11/28/16

This week, Yi and I a pretty hands-on lab day. We performed out mini experiment involving different solutions of protein, urea, and buffer to measure how concentration of urea affects protein folding. After combining a solution of buffer and protein with another solution of buffer and urea, we used advanced spectroscopy to measure the intensity of proteins in the folded versus unfolded proteins at different concentrations.

I had to make a table of expected molarities, prepare the two different solutions, and combine the solutions in the appropriate ratios to perform the spectroscopy. Making the table of expected molarities was pretty simple, as Yi told me what the total solution volume should be (120 microliters) and what the expected urea concentration was. From there, all it took were a few molar conversions to figure out how many microliters of each solution were necessary. Below I have recorded the table of expected molarities.

Concentration (M)	Volume of protein + urea (µL)	Volume of protein + buffer (µL)
0	0	120
.2	6	114
.5	15	105
.5	24	96
1	30	90
1.2	36	84
1.4	42	78
1.7	51	69
2	60	60
2.3	69	51
2.5	75	45
2.7	81	39
3	90	30
3.3	99	21
3.6	108	12
4	120	0

Once the table was finished, we had to prepare the solutions. I used a scale to measure the proper amounts of urea to add to the protein solution, using molar mass to determine the number of grams of urea necessary for creating the proper concentration of solution. We then mixed the predetermined volumes of urea/protein solution with the buffer/protein solution to make the necessary concentration in twenty different vials. It should be noted that the amount of protein was constant in each vial, because our independent variable was urea concentration, not protein concentration.

We then place the vials of varying solution into the spectrometer to get a measure of protein absorbance under different urea concentrations (and thus proteins in different folding states). It took awhile for us to get results for our mini experiment because the spectrometer had to be warmed up and calibrated for the light intensity we were using. Unfortunately, I could not download and print the results of our experiment, but I can say that the peak absorbance lay within the middle range of urea concentration, after the pH was too basic but before it became too acidic and the protein denatured.

Although our experiment was brief and not very official,it was great practice in the lab and gave a good overview of the equipment and techniques we will be using in the future!

Week of 11/14/16

This was my first week back since October. As explained earlier, I was in New York City a few weeks ago on an opera trip, and last week I had to attend an unexpected funeral. Needless to say, I was very excited to get back to work at RPI. Although my visit this week did not go as expected, it was enjoyable nonetheless.

Yi had a big presentation to give after our visit on Tuesday, so we didn't have enough time to visit the lab this week. Instead, we sat in his office and he walked me through his PowerPoint, which he will present at a large conference in New Orleans in a short time. Before you speculate that this week was wasted, I will assure you that his presentation was very much related to what we are working on.

In his experiment, Yi used HSQC (heteronuclear single quantum coherence spectroscopy) and high pressure NMR (nuclear magnetic resonance spectroscopy) to investigate the relationship between pressure and protein states. Essentially, he was looking at the behavior of folding proteins under different pressures. Do more proteins enter the folded state under really high or really low pressures? Generally, Yi found the answer to be somewhere in the middle. Moderate pressures tended to work best for proteins in order for them to fold and take shape. Pressures that were too high or too low seemed to cause the folded proteins to denature and remain in the unfolded state.

I do not have a picture of Yi's presentation, but I have pictured below a sample NMR graph as well as a few images that illustrate that ideal pressures for protein folding lie not on the minimum or maximum extrema, but within the middle range of the pressure values.The wavelength with the highest intensity of folded proteins correspond to mid-range pressures.

NMR intensity image. Source: http://www.cbs.cnrs.fr/index.php/en/research-equipe1/rmn-haute-pression

This week did not go as expected, but it was really fun! I really appreciated Yi's willingness to share his presentation with me. He will do great in New Oreleans. Until next time!

Week of 10/31/16

I was in New York City with the choir this past Tuesday. We got to see an amazing opera, and I had so much fun. Unfortunately, this meant that I could not attend my internship. Because we did not meet, I decided to take a few minutes once we returned from the trip to gather resources for my amino acid studies. The most useful diagram I found is shown below.

Amino acid study chart. Source: https://en.wikipedia.org/wiki/Proteinogenic_amino_acid#/media/File:Molecular_structures_of_the_21_proteinogenic_amino_acids.svg

I also love Leah4sciMCAT's videos.She has clear, concise presentations on amino acid structure, function, and properties. Here is a sample video about basic structures of amino acids.

Amino acids introduction to structure charge classification and reactions video.
Source: youtube.com/leah4scimcat

All of these resources make it so much easier to study amino acids. Thank goodness for modern technology! Can't wait to get back to my internship next week.

Week of 10/24/16

This week was fun! Yi didn't have any specific plans in the lab today, so we made plans for a mini experiment that involves measuring protein fluorescence (and by extension, structure) when the protein is exposed to different levels of urea. Urea is an organic compound that plays important role in the metabolism of nitrogen-containing compounds. It is used in many bodily processes, particularly involving nitrogen excretion.

Before beginning our work, we had to isolate a protein sample from a protein-buffer sample. To do so, we used a centrifuge. A centrifuge is a machine with a rapidly rotating container that applies centrifugal force to its contents, typically to separate fluids of different densities. When the centrifuge is finished its cycle, the protein and buffer solution separate, leaving an isolated layer of protein that can be in an experiment. A detailed picture of how this machine works is below.

Centrifuge diagram. Source: http://cdn4.explainthatstuff.com/centrifuge-high-speed.png

Once we had our protein sample isolated, we had to make a plan as to how we would alter the concentration of urea in each sample we will use to examine protein fluorescence. The ratio of urea to buffer needs to increase each time. But first, we needed to make the stock concentration of urea to be diluted. We made a solution of buffer and urea, and I had to do all the molar conversions and manipulations to translation the number of grams per milliliter of each substance.

Once the calculations were done, we massed out the number of grams of urea and CC necessary and combined them in the buffer solution. Urea takes a very long time to disolve in water, so we placed our mixture in a warm water bath and shook the combination jar very vigorously. By the time the urea had started dissolving, our time was up! Yi had a chemistry lecture to get to and I had a transport van to catch. This week was a fun one, and I am so excited to go back. Until next time!

Week of 10/16/16

This past Tuesday, I visited RPI for my first lab session! I received my own official badge that allows me access into the CBIS building and laboratories. This was a cause for great celebration for both me and Yi, the graduate student I am working with (who no longer has to race to meet me at various entrances when I call to be allowed entry into the building).

Besides receiving m own visitor's badge (and a pretty rad pair of safety goggles), Tuesday was the first day of official (ish) lab work for me. Yi took me around the lab, giving me a tour of his designated station and all the equipment we will be using throughout this process. I discovered the centrifuge, uber-fancy pipettes, protein fridge, and more. Once I was familiarized the area and debriefed on laboratory safety, we began working.

Since it was my first official day, Yi wanted to make sure I understood the basics of lab techniques we will be regularly employing during our work this year. We talked about calculating protein concentration using absorbance, the molar absorptivity constant, and path length (hello, Beer's Law!). Essentially, calculating the protein concentration can be boiled down to dividing the absorbance by the absorptivity coefficient (m.a. constant x path length). I was really happy to discover that I already knew and understood what Yi was explaining to me before he finished, and his recap of Beer's Law was a great refresher. We were both (at least, I think it was both of us!) pleased to realize that my science background had prepared me well for this experience, and it was time for lab work to begin.

I participated in a simple spectroscopy demonstration under Yi's supervision. We took a small amount of DI water and placed it onto the slit of the spectrophotometer, Then, we closed the machine and allowed it to run. This set the blank for the machine. Next, we loaded a small amount of our protein sample onto the slit, and allowed the machine to run. Eventually, the computer relayed the result of the concentration of our protein sample. How does a spectrophotometer work, you ask? Here is a simple picture that explains, though ours did not occur within a cuvette.

Spectrophotometer diagram. Source: http://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry

After the lab seesion was done, I went back to Yi's office and watched videos about amino acids. I need to be easily familiar with the different groups- knowing their structures, properties, and names will be crucial for this experiment. I've continued my exploration of amino acids this past week, and hope that I will be a pro on all the R groups this time next week. Wish me luck studying!

Week of 10/09/16

My first post! This year I am taking part in the CBIS High School Scholars Program at Rensselaer Polytechnic Institute. I will be interning under Dr. Catherine Royer at the Center for Biological and Interdisciplinary Sciences (CBIS) at RPI, and working on protein purification. Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues, or whole organisms. This process is necessary for characterizing the structure, nature, and interactions of a selected protein.

Protein purification flowchart. Source: http://2009.igem.org/Team:Washington/Project

This past Tuesday our introductory meeting took place. I met with Mr. Calos, Dr. Royer, and Yi, the graduate student I will be working with, to discuss the meeting times a procedures for this year-long internship. We discussed my background in science, how laboratory proceedings will occur, and the expectations set for me as my senior year (and this internship) continues. I found out very quickly that I really enjoy the company of both Dr. Royer and Yi, which only makes me want to succeed even more. Hopefully, I will be able to put in enough time, effort, and enthusiasm to make this internship a success. Until next time!