4/10-4/16: Week 9 – Entering the Wet Lab

                             

This week I finally entered the Mills Lab's wet lab and began working on research alongside a student pursuing his master's degree, Anthony (Tony) Meza.

Tony's research focuses on designing a functional metalloprotein which incorporates the non-canonical amino acid (not occurring in nature) containing bipyridine as its side chain. This research may lead to the development of protein assemblies with photochemical and photophysical properties that would be difficult to achieve with standard amino acids.

To accomplish this goal, several plates of mutated and wild type (non-mutated) E. coli bacteria were grown and thereafter harvested. Using the harvested cells, I used a polymerase chain reaction (PCR) kit to rupture the cells and thereafter induce an artificial, exponential DNA replication process.

Nano drop I used to measure concentrations of E. Coli DNA. By understanding the concentration and size patterns, I was able to identify the best samples to use for the PCR.

Tony's rack of primers and E. coli genetic material from various cultures awaiting replication.

While working on the project, I also got to learn about many tangential topics, such as the dangerous nature of ethidium bromide and how it can kill cells. I also learned that the LD50 (the dosage of a substance that kills 50% of a test population) for orally ingested caffeine is between 150 and 200 mg/kg for an average adult, or roughly 80 to 100 cups of coffee.

The molecular structure of ethidium bromide (EtBr), which is utilized as a nucleic acid stain in gel electrophoresis. Just like all other polycyclic aromatic hydrocarbons (hydrocarbons with several aromatic rings in their structures), EtBr is thought to be carcinogenic because of its ability to be incorporated into DNA. However, Tony explained to me that EtBr actually prevents DNA replication and therefore is actually cytotoxic rather than carcinogenic. There has also been no significant evidence for EtBr's supposedly carcinogenic nature. This isn't important to my research in the lab as EtBr is still dangerous even if it isn't carcinogenic, but I thought that this was an interesting tidbit. 

Loaded agarose gel in a buffer solution. The pink well holds the "ladder" (metric) and the green wells hold what I hope to be concentrated E. coli DNA. Running an electric current through the gel and surrounding buffer will cause the DNA to migrate through the porous gel. This is due to DNA's intrinsic negative charge, which attracts it to the positive end of the plate while also repelling it from the negative charge created by the current.

As seen in the image, the green food dye has decayed into its components: blue and yellow. This is due to the two dyes' different interactions with the current. These dyes do not matter to the end result of the gel electrophoresis as they were simple visual markers that don't bind to nucleic acid. The EtBr, on the other hand, bound to the nucleic acids in the wells will create patterns parallel to the ladder, thus identifying the molecules by their sizes, which determine how far each molecule migrates. I'll post the result in my next update this coming week!

See you next week!

3/26-4/09/2017: Weeks 7 & 8 – Preparing for Wet Lab Work

So, at long last, I turned 18 at some point in the past two weeks. This means that I am finally able to begin working towards working in the Mills Lab’s wet lab at the ASU Biodesign Institute. In order to prepare for my position, I’ve spent the last few weeks studying lab safety manuals from my previous labs in addition to completing the courses required of me from ASU for me to be able to work independently in a lab. The courses and exams I’ve taken in these efforts include topics ranging from Biosafety to Fire Safety.

In the courses I took, I re-learned about how to properly dispose of the remnants/results of my research in a safe and careful fashion. The detailed descriptions of the 4 biological safety levels and the proper actions which ought to be taken in the case of each further emphasized the variety of dangers and situations which may arise over the course of scientific and especially biomedical research.

This coming week, I will be taking an in-person laboratory safety class and also receive a badge which will grant me access to the ASU Biodesign building. Once I complete these steps, I’ll be able to smoothly transition into the wet lab from bioinformatics.

See you next week!

3/18-3/25/2017: Week 6- Combining the Molecular Modelling Softwares' Benefits into a Powerful, Revised Foldit

So this week, I took some time to confirm my research into each of the three 3D modelling softwares which I have been using for molecular and protein modelling. Afterwards, I grouped the benefits of each program so that I might better lay out a road map which the Rosetta Commons may follow to modify their Foldit product so that it may be more multifaceted and applicable to a wider variety of circumstances.

Additionally, I considered some other features which I'd add given the release of the Foldit source code.

Foldit base

•3D modelling

•Default ribbon structures

•Solid molecular shapes (smallest manipulable units are amino polymer side chains)

•Collision-elimination algorithms

•Hydrophobic/hydrophilic molecules are labled

•Hydrogen bones are illustrated and their importance to protein synthesis is aptly demonstrated

Introductory protein design features

Avogadro

•Molecule construction

•Import functionality

•Range of modelling goes from atoms to full-scale molecules

•Automatic geometry correction algorithms

•Detailed imaging

•Implement the range of modelling to include Foldit ribbon structures and Avogadro ball-and-stick model via simple if statement.

PyMol

•Command line features with easy access to computer files

•Replace tool bar in Foldit

•Frame –by-frame move/playback feature and snapshot command

•Leave the UI in favor of a more aesthetically appealing one such as Avogadro or Foldit

•Combine the ideas of automatic molecule to atom viewing originating from Avogadro via simple magnification threshold addition and resulting shape change to PyMol’s Python code.

Additional features which may be added

•Pre-constructed cellular structures, such as ribosomes, with automatic re-focus feature. Since the programs deal with molecular and protein design, inserting a body hundreds of times larger than an amino acid would make the model unmanageable without auxiliary functions which automatically adjust the magnification and label all structures smaller than a few nanometers.

•An auto-play feature in which the interactions between two selected structures may be demonstrated. For example, if an amino acid chain and a ribosome are selected, the ribosome constructs a protein by accessing an online library via looking up the amino acid chain that’s entered into the ribosome. Thus, the process of protein synthesis may be illustrated with great detail. However, this would take a great deal of time and coding which may only be made available should the Rosetta commons allow access to the Foldit source code.

3/03-3/11/2017: Week 4 – Studying the Full Capabilities of Multiple Molecular Modelling Softwares

This past week has been very useful in terms of gaining knowledge on the workings of the PyMol and Avogadro programs. Unfortunately, I would only be able to share this information with you through an absurd amount of function definitions and pictures, which I shall not bore you with.

However, there are two features which I am particularly excited about. One is the molecular geometry optimization function of Avogadro. In manually building or even editing uploaded molecules via the UI, it’s possible to utilize the Optimize feature so that the molecular geometry is corrected to reflect the laws of chemistry. This feature touches every aspect of the molecule short of its atomic pairings.

The second feature which I found is PyMol’s “movie” feature. This part of the program allows for an addendum to the load command which gives the option for multiple files to be loaded in reference to one another as different frames of a single “movie” or animation. By the syntax of load [file directory-specific name], [selection name], [state or “frame”].

Additionally, this week I found that both Avogadro and PyMol are both capable of loading .pdb files. This revelation makes a batch script I’d developed specifically for the purpose of automating the transferring Avogadro-created files to the PyMol environment totally useless. On the bright side, I got to study batch some amount and that was a rather entertaining adventure in itself.

This current week, I am beginning to work on my own archetype for the Foldit module by describing the functionalities I envision would be included in it as well as illustrating the sorts of coding methods I see being implemented to expand the platform’ uses.

See you next week!

2/20-3/03/2017: Weeks 2 & 3 – Understanding 3D Molecular Modelling

Since my initial post and abstract about my project, some changes have arisen. Firstly, rather than actually editing the source code of Foldit, I will be instead constructing an archetype for any future versions. This change was brought about by issues in obtaining access to the source code from the IP license holders, the Rosetta Commons.

Instead of straightforwardly working with Foldit, I will be using to other programs, PyMol and Avogadro, to design a system akin to what I’d like to add to Foldit. Avogadro is an advanced molecule editor and visualizer with very high processing capabilities. PyMol is an older software which depends on the Python language for its structure and functions. This feature allows it to have a command system based around the language. However, the learning curve for this command system is fairly steep and the user interface is nowhere nearly as intuitive as that of Avogadro’s.

The results of me attempting to better understand Avogadro (right) and PyMol (left) by inserting similar atoms/molecules in either program.

I still have much to learn about PyMol and Avogadro but I have been making steady progress with both programs. Next week I plan to utilize the knowledge I am continuing to gain in these programs to begin developing ways to take advantage of the respective benefits of PyMol and Avogadro.

At the moment, I envision this combined system to work by the initial molecules being constructed in Avogadro with its relatively simple building mechanisms, after which the molecules would be saved as perhaps .xyz files and thereafter loaded into PyMol. The purpose of .xyz files is to contain the x, y, and z coordinates of the data contained within the file. This extension is perfect for 3D modelling. Upon importing the file into PyMol, the file will be converted as need be to fit PyMol’s specifications and thus the molecule will be open to edits by manual interactions and commands in the UI. The specific features of this process is what I'll be working on now.

See you next time!

2/13-2/19/2017: Week 1 – Background Information

Hello! In my first week of working on my senior research project, I found it important to educate myself on the topic which I will be working on in addition to formulating my project’s timeline and end goals.

ABSTRACT

Metalloproteins are proteins that contain a metal ion cofactor and compose between a quarter and half of all proteins in the human body. This metal cofactor typically dictates the protein’s function and influences a protein to complete tasks varying from oxygen transportation to signal transduction, like “fight or flight” reflex. In addition to making up a significant amount of the body’s proteins, metalloproteins are relatively easy to design and implement in biological/cellular models. The Mills Lab at ASU Biodesign aims to increase our understanding of metalloprotein functionality and further develop these biological “worker” molecules to broaden their applicability. This research works to form specific molecules and analyze the structures and functions of the created compounds. To provide further education on proteomics and amino acids for the next generation of scientists, the lab is also developing a supplemental educational tool for biology students using Foldit software.

METALLOPROTEINS

Metalloprotein functions are encoded by the metals which are bound within their primary structures. These proteins are relatively easily designed and can be easily thrown into cells to degrade toxins and small molecules (approx. 50-100 atoms) and biopolymers (i.e. proteins). Through the metal cofactors in metalloproteins, they can be used as programmable catalysts in abiological contexts, leading to extrasomal protein utilization. These implications extend beyond poison/toxin removal to material design.

FOLDIT

Foldit is an online puzzle game about protein folding. It was developed by the University of Washington, Center for Game Science, and the UW Department of Biochemistry. The objective of Foldit is to fold the structures of selected proteins using tools provided in the game. The highest scoring solutions are analyzed by researchers, who determine whether or not there is a native structural configuration (native state) that can be applied to relevant proteins in the real world.

QUESTIONS

Can we successfully mutate metalloproteins while predicting their resulting functions?

How can we examine our results to ensure that our desired results are achieved?

What steps must be taken to produce our desired protein structures?

How can we modify Foldit to facilitate education in biology and proteomics?