Week 3 Problem set

PSET #3

ASSIGNED: Friday 24 April 2020

DUE: 5:00p Pacific 01 May 2020

NOTES:

Given the unique circumstance of Spring 2020, we ask you to do your best to maximize your learning. Each problem set is an opportunity to assess your learning, identify gaps, reflect on what you have learned, and determine what you wish to learn next.
Problem sets must be completed individually unless stated otherwise.
Please turn in your completed problem sets as an electronic copy via Gradescope. Please make sure to clearly indicate the starting and ending boundaries of your answers to each question on Gradescope.
Please do not go over any word limits and where appropriate show your work (e.g., calculations with appropriate units).
Please type your answers when possible.

(Q1) Revisiting Design Tools (PyMol) (30 pts)

In this problem, you will use PyMol to visualize green fluorescent protein (GFP). GFP is a 238 amino acid protein. For more information about GFP, check out the Wikipedia page, or see the resources below.

Check out the website where the GFP structure is housed: https://www.rcsb.org/structure/1gfl. Notice that we are looking at a homo-dimer, meaning that 2 identical 238 amino acid chains are in the structure.

Note: Please include figure labels when sharing figures.

1.a. What is the experimental method used to get the structure of the protein? What is the listed resolution of the structure? (bullet points)

1.b. Add a screenshot of your GFP structure in PyMol showing the “beta barrel” structure of fluorescent proteins.

To do so:

Download PyMol from this Pymol Link and open PyMol on your laptop or computer.
Type in the command “fetch 1gfl” in PyMol. This command downloads the structure of Green Fluorescent Protein (GFP) from the Protein Data Bank (PDB)
In PyMol and take a look at the GFP structure. Under the menus “H” and “S” listed near the structure name on the toolbar on the right, click Hide everything and Show cartoon.
Take a screenshot of the protein structure to share as part of the problem set

Extra activity-1

Click on <Mouse → 1 button viewing> to enable scrolling.
Click on <Display → Sequence On> to see the amino acid sequence for GFP.
Select residues 64-66 SYG and color these residues red. This is the chromophore of GFP, the portion of the molecule responsible for the fluorescence.
Render the SYG portion of the protein as ‘sticks’. You should now be able to see the (cyclized) ring of the GFP chromophore. Take a second screenshot of the protein with chromophore highlighted.

1.c. Go back to the Protein Data Bank website and find a .pdb file for another fluorescent protein. Use PyMol to visualize this second protein. Provide a screenshot and tell us what color and wavelength the protein fluoresces. Briefly compare the similarity/ differences in the structure of the two fluorescent proteins (yours vs. GFP).

Extra activity-2 Would you like to build your own 3D GFP paper model? Go to the following link and use the instruction and the template to build your own model. If you did make the model please share a photo with us.

Additional Resources (Fluorescent Proteins): Fluorescent Proteins and the Story Behind GFP (by Roger Tsien ibiology) link-1 Introduction to Fluorescent Proteins link-2 Interactive graph describing Fluorescent protein properties link-3

(Q2) Foldscope (40 pts)

2020 Note: Given the unique circumstance of this quarter and if you don’t have access to your own foldscope you can use the photos available from Microcosmos. For example, see an entry from last year’s BIOE80 class: link.

The following is an exercise carefully designed by Tom Knight to help you understand if you are more interested in science or engineering. There is no right answer and all types are welcome in the course! Rather, what you experience via the following can help you (and us) understand how you most like to work and learn. I.e., what follows is analogous to learning which foot you prefer to put forward on a skateboard, surfboard, or snowboard (right of left), or whether you prefer to throw with your right or left hand, etc.

Self-Assessment:

Imagine that you spend hours assembling a device (a box) that, once finished, is supposed to power a light bulb when a button is pushed. You place your finished construction on your workshop table and press the button… POOF! Smoke pours from the device but the bulb remains dark.

Do you exclaim (choose one):

      DARN!!! 	COOL!!!

2.a. Why did you pick your choice? (1-2 sentences)

Relax again. Close your eyes and look inside yourself. Relax some more. Might you want to be an Explorer of the Unknown? Or, a Maker of New Things? Choose one path to follow for now. Focus on one path for now so that you can really push yourself in the context of this activity. Use your answer to Step 1 to think about your choice.

If you picked DARN!!! please go to the Maker step.

If you picked COOL!!! please go to the Explorer step.

2.b. Welcome Explorers!

You have been granted the status, biologically curious explorer of the microcosmic universe! Your job is to explore objects, mysteries, and open questions that relate to the microscopic world. Equiped with your own microscope.

Firstly, you have to ask a question in the realm of micro-cosmos, an original question. It does not matter if somebody has answered it 100 years ago or if science does not know how to begin to think about it. Choose a question that can be explored with microscale observations. Such as - how many cells thick is a leaf, how do hairs split, do insects also have blood cells, what does insect skin look like, what mold is growing in my fridge, what grows in a drop of pond water…You get the idea. Now based on your emerging question, collect samples and visulize them with your microscope.

To complete this activity, prepare and submit the following entry (1 paragraph).

Consider including responses to the following items below. (If you use a photo from microcosmos instead of your own photo make sure to reference the source).

What question or what range of questions did you ask?
What did you see / find as a result?
A great image or data to support your findings and conclusions (Note for 2020, photo does not have to be your own).
Your concluding thoughts or open questions.

2.b. Welcome Makers!

You have been granted the status, biologically empowered tinkerer of the microcosmic universe! Your job is to explore nature, searching for tiny objects that might be useful either as inspiration, or as bits and pieces that might be directly reused to make something.

Firstly, you have to learn to explore various realms of microcosmos. It does not matter if somebody has searched the same space 100 years ago or if engineers do not know how to begin to think about the tiny things you find. Hunt the patterns, solutions, and objects that can be explored with microscale observations. Such as how do plants transport water, how do grasshoppers hop without barrel rolling, how does a spider make a web, where do leaves go… You get the idea.

Your goal is to go on a journey to find the answer yourself, without any other aids. Lots and lots of samples that could give you the diversity of answers you might be looking for.

To complete this activity, prepare and submit the following entry (1 paragraph).

Consider including responses to the following items below. (If you use a photo from microcosmos instead of your own photo make sure to reference the source).

What useful biological object or potential inspiration from nature did you find?
How and where did these samples were found?
A great image or data to support your findings and conclusions (Note for 2020, photo does not have to be your own).
Your concluding thoughts or open questions.

Extra activity-3: Submit a photo you have taken with Foldscope to Microcosmos and share the link with us as part of PSET

(Q3) Papers: KumaMax & Repressilator (15 pts)

In the pre-class material you were introduced to KumaMax designed via the student iGEM project. The following link will take you to the paper that the group published summarizing their efforts, Computational Design of an α-Gliadin Peptidase

3.a. In your own words, what is the primary claim of the paper? What are the primary evidence in support of the claim? (2-3 sentences)

Next, visit the famous Repressilator paper, A synthetic oscillatory network of transcriptional regulators.

3.b. In your own words, what is the primary claim of the paper? What are the primary evidence in support of the claim? In your own words, describe how does the Repressilator work? (2-3 sentences)

3.c. Are all cells oscillation together (i.e., turning bright in synch. with one another)? Why not? (2-3 sentences)

Extra reading: A review titled, Synchronous long-term oscillations in a synthetic gene circuit

(Q4) Prepare for the Group Project (15 pts)

Kiva is a charity that has a unique approach to the alleviation of wants and needs.

Instead of soliciting donations, Kiva provides a platform for individuals and groups around the world to request loans for their cause, empowering them to build a better future for themselves, their families, and their community.

Take a few minutes to browse the Kiva website, noting the geographic location of the groups you see and their goals.

Note that you can select various sectors (health, food, agriculture, arts, education, … ) or locations across the world.

4.a. What is your initial response to the Kiva platform? (1-2 sentences)

Kiva breaks down into different topics, allowing the potential lender to filter by the topic they find the most compelling. Looking at a few of the most common loan requests and we can extract a general structure:

Campaign...	Goal / Use of Loan...
Cruz Celina, Hùng, Loan	Buying materials to build a latrine
Channy's Group, Sreymai’s Group	Buying water filters
Jane’s Group, Sin Kyoe(2)D Village Group, Zulaykha	Buying fertilizer, seed
Kantiembou Group, Dumitru, Glenda, Zhanybek	Buying livestock

This focuses on solving problems immediately by providing (consuming) products as they currently exist. In this class, we have the opportunity to step outside the bounds of current offerings and thinking about designing and building better solutions that fix unmet needs.

4.b. Name 3 sectors that are of interest to you?

4.c. What role, imagined or as yet unimagined, can bioengineering play in addressing the needs in these sectors? (2 to 3 sentences)

4.d. Pick a location (ideally a region that you are not familiar with). What are 3 to 4 specific requests that people from this location have?

4.e. What role, imagined or as yet unimagined, can bioengineering play in addressing these requests? (2 to 3 sentences)

(Added to turning in the answers as part of the PSET, have them ready as “introduction cards” to exchange ideas with your future team)

Extra resources, learning, and Practice Questions

The following suggested reading and questions are 100% optional additional. They are meant to help you engage with various aspects of the topics that we have covered in each segment based on your interest and level. They are not required as part of your problem sets. But we hope that they enable you to enhance your learning based on your interest.

Optional video lectures from Protein design by David Baker

Q.EX1. Practice Questions: Physics of Fluorescence (0 pts)

This question is a guide to enable you to learn (or review) some key physics of how fluorescence works. Use the following resources to familiarize youself with physics of fluorescence: Hyperphysics-link Thermo fisher-link

The following equation relates a photon’s energy to its wavelength:

$ E= \frac{hc}{\lambda} $

E is the energy of the photon (in electron volts, eV, or in joules, J),
c is the speed of light (in meters per second, m/s),
h is the Planck constant (equal to 6.626×10^−34 J*s, with units of energy times second),
and $ \lambda $ is the wavelength of the photon (with units of length, in nanometers, nm).

EX.1.a What happens to the energy of a photon as the wavelength of light increases? (Extra activity demonstrates to yourself that eV and J are equivalent to each other?)

EX.1.b If a traveling photon hits a GFP molecule, the probability that the photon gets absorbed by GFP depends on the wavelength of the photon. In a sense, GFP “prefers” photons around a certain energy level. If GFP absorbs a photon, it enters a higher energy excited state for a brief period, then relaxes back to a lower energy state. The absorbed photon’s energy is converted into the jiggling of the GFP molecule, heat that the GFP vents to its environment, and a photon that gets emitted from the GFP. The wavelength of the emitted photon depends on how much energy is converted to jiggling and heat versus how much energy is converted to an emitted photon, which is determined somewhat randomly.

The most probable wavelengths of absorbed and emitted light respectively, are the “excitation” and “emission” peaks. Typical excitation and emission peaks for GFP are reported below. What are the energies of photons typically absorbed and emitted from GFP?

GFP	Wavelength (nm)	Photon Energy [unit]
Excitation Peak	395 nm	? [unit]
Emission peak	509 nm	? [unit]

EX.1.c What is the difference in the wavelength between the emission and excitation peaks of GFP? What is the difference in energy between photons at these two peaks?

EX.1.d Does it make sense that the wavelength of an emitted photon is longer than the wavelength of an absorbed photon? (Yes/No) Why? (Use the calculated numbers from part B in your answer).

EX.1.e You are studying how GFP absorbs and emits light when you notice something odd. Under certain conditions, GFP seems to have a smaller excitation peak around 800 nm. Why might this be the case?

Q.EX.2. Foldscope Engineer (0 pts)

After finishing BIOE.80 you have joined the team that develops Foldscope.

First and briefly familiarize yourself with this open source scientific paper summarizing various aspects of the Foldscope. Briefly read the abstract and go through the Figures 1-5 and Table-1.

EX.2.a What is the main claim of the paper based on the abstract? What key evidence is provided in support of this claim? (Bullet points.)

Next, your new manager shares the following equations describing the optical properties of a ball lens from the paper’s supplementary material.

$ M=\frac{250mm}{EFL} $

M is the magnification,
EFL is Effective focal length,
250mm is the distance between the lens and where the image forms.

Additionally:

$ EFL=\frac{nr}{2(n-1)} $

r is the lens’s radius,
n is the index of refraction

You want to create a figure to communicate to your team the relationship between M as you change the radius of the lens.

Assume the index of refraction for the borosilicate glass lens is n =1.517. Also, assume that you can purchase glass lenses ranging from 150, 250, 400, 750, and 1000 µm in radii.

EX.2.b Using your favorite software/application (e.g., Spreadsheets, Matlab, Python, R, etc.) create two graphs demonstrating the relationship between EFL vs. r, and M vs. r. Make sure to label each figure and each axis with appropriate units.

EX.2.c Next, state your conclusions from these graphs summarizing your findings in 1-2 sentences to the rest of your team.

EX.2.d Next, you come across sapphire lenses, with n=1.77. Using the equations above, compare the Magnification of a Sapphire lens against a borosilicate lens. (Hint: Assume that both lenses have the same size)

EX.2.e As you are going through this design activity, a team member asks you about the difference between resolution and magnification. First, Describe the difference between resolution and magnification and include your reference(s). (Hint take a look at figure-4.b from the paper, describe the relationship resolution and magnification)

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