Friday, March 31, 2017
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!
Sunday, March 5, 2017
Week of 3/5/17
Yi had a meeting today so I couldn't get to RPI, but I eagerly anticipate our next session!
Wednesday, February 8, 2017
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!
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!
Monday, January 30, 2017
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!
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!
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.
Monday, January 23, 2017
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!
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!
Tuesday, January 3, 2017
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!
Sunday, December 11, 2016
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.
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!
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