Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes, Final Report

This summer, Arianna, Dr. Chang and I researched the topic of Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes. Our hopes were to learn about the self-assembling properties of silicateins and use this information to develop new forms of cathepsin-L to adopt these properties. This would involve determining which amino acid residues are responsible for silicateins self-assembling properties and  then making changes to amino acids within cathepsin-L.

The beginning of our summer was spent getting Arianna and I comfortable with using various lab equipment. We prepared materials such as antibiotic-containing plates, chemical transformations with e. coli, and mini-preps towards DNA analysis. These steps aided in learning how to follow procedures accurately. After these preliminary procedures we took some time to do computational work and create our poster which was presented at the 2018 Gordon Research Conference on Biomineralization, held at Colby-Sawyer College in New London, New Hampshire from July 28 – August 3. My section of the poster involved searching for possible future uses of biosilica prepared with silicatein. I used Web of Knowledge to search through literature and found that biosilica has potential to be used in regenerative medicine as well as 3D printing. After some research of my own I came to the conclusion that there is very limited information focusing on future uses of silicatein, and I am curious as to why this is.

Initially, some techniques we were supposed to learn included: protein mutagenesis and characterizing hydrophobic and electrostatic protein-protein interactions. Tentative methods were: purifying, manipulating and characterizing proteins; forming and refining silica mineralization assays in the absence and presence of proteins; completing scanning and transmission electron microscopy, atomic force microscopy, focus-ion beammilling, and finally, analyzing complex datasets and interpreting the results. Unfortunately, we did not get to complete as much as we wanted due to unsuccessful protein-expression systems.

We started with trying to express the protein lactate dehydrogenase from the Mackerel ice fish (c. gunnari). The first system used a pUC57 vector purchased from GeneWiz containing the cgLDH gene. The goal was to express the cgLDH gene in the DH5-ɑ e. coli strain purchased from New England Biolabs. This attempt was unsuccessful despite the vector’s compatibility with the DH5-ɑ e. coli cells. The system was not able to produce and isolate our protein. For our second attempt, we used a pET11a vector with the cgLDH gene inserted purchased from GeneScript and T7 Express e. coli from New England Biolabs. Using a NanoDrop 1000 spectrophotometer we once again measured no protein present. After performing a more sensitive assay, Dr. Chang concluded we produced the desired protein, but in very small quantities. I would like to find out why we are not seeing the amount of protein one might expect, and which step of the system is hindering production.

I believe our next step is to modify our procedure to optimize growth conditions and produce the protein in large quantities. The expression of cgLDH is important for a research project that is in collaboration with research groups at Albert Einstein College of Medicine. This means that even though cgLDH is not directly related to our current summer research project, we hope cathepsin-L can be expressed using T7 Express e. coli and the pET11a vector as well.

This is my first time working in a research lab, so each step along the way has been a new opportunity for me to learn. From the first day learning how to use the autoclave, to our last meeting when we went through an entire protein-expression system, I have learned a wealth of new lab skills. Exposing myself to new academic experiences and growing as a student as well as a team member are the key things I would like to achieve from this project. Through our research, I hope to expand my knowledge of proteins, enzymes, and the amino acids which are the building blocks behind them.

I have certainly learned some important lessons from this project. I have learned that in research there is a lot of trial and error that goes on, and sometimes you can work for hours or even days on a single project and have it be unsuccessful. I’ve learned from Dr. Chang’s example that you can’t let these setbacks throw you off or deter you from trying a new procedure. I’ve also learned that it is important to use your resources and have those who are experts with certain materials help you if you are unsure where the process is failing. Our experience this summer leaves me determined to perfect our protein-producing system in the future so we can continue with our project.

 

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Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes, Blog Post #2

We are still in the initial stages of our research, which means we have not yet perfected our systems and procedures. Arianna, Professor Chang and I spent time working on a poster which was presented at the 2018 Gordon Research Conference on Biomineralization, held at Colby-Sawyer College in New London, New Hampshire from July 28 – August 3. My section of the poster required me to search for possible uses of biosilica prepared with silicatein. To do so, I read through literature found during our search using Web of Knowledge. I learned about the possible uses of biosilica in fields such as regenerative medicine, as well as 3D printing. I also came to the conclusion that there is not much information out there on our specific studies which limited the contribution of future uses I could add to the poster.

As far as lab work, we have worked on creating a successful method in which we can express protein. Along the way we ran into some issues. Our group started with trying to express a protein called lactate dehydrogenase from the Mackerel ice fish (c. gunnari). Our first system included a pUC57 vector purchased from GeneWiz containing the cgLDH gene which we hoped to express in the DH5-ɑ e. coli strain purchased from New England Biolabs (NEB). Unfortunately, we were unsuccessful this attempt, due to the fact that the construct was not compatible to express our cgLDH protein. For our next attempt we used a pET11a vector with the cgLDH gene inserted from GeneScript and T7 Express e. coli from NEB. When collecting data at the end of our expression system, we initially thought we had once again not produced any protein because measurements using a NanoDrop 1000 spectrophotometer implied protein was not present. After Professor Chang performed a more sensitive assay, he concluded we did indeed produce the protein, although in very small quantities. I am curious as to why we are not seeing the amount of protein one might expect, and which step of the system is hindering this production.

I believe in the future we need to modify our procedure in order to optimizing growth conditions to produce the protein in large quantities.The expression of cgLDH is important for a research project that is in collaboration with research groups at Albert Einstein College of Medicine. Thus, even though cgLDH is not directly related to our current project, it is in our hope that cathepsin-L can be expressed using T7 Express e. coli and the pET11a vector as well.

I have certainly learned a few lessons from this project. I have learned that in research there is a lot of trial and error that goes on, and sometimes you can work for hours or even days on a single thing and have it be unsuccessful. I’ve learned from Professor Chang’s example that you can’t let these setbacks throw you off or deter you from trying a new procedure. I’ve also learned that it is important to use your resources and have those who are experts with certain materials help you if you are unsure where the process is failing. Our experience this summer leaves me determined to perfect our protein-producing system in the future so we can continue with our project.

 

Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes, Blog post #1

The topic Arianna, Dr. Chang and I are researching is Iterative Site-Directed Mutagenesis Towards the Directed Evolution of Enzymes. The purpose of this project is to understand the self-assembling properties of silicateins and try to develop new forms of cathepsin-L which will adopt these properties. To accomplish this, we must determine which amino acids residues are responsible for the self-assembling properties of silicateins. Once we have this knowledge, we will make changes to different amino acids within cathepsin-L. Ultimately, the goal of this research project is to aid future studies in utilizing the self-assembling cathepsin-L to create materials for the use in medical implants and optical materials.

So far, our time spent in the lab has been focused on preliminary preparation of materials such as antibiotic containing plates, chemical transformations with e. coli, and mini-preps towards DNA analysis. We have also spent some time conducting a literature search using Web of Knowledge. We researched relevant literature published in recent years that could help us throughout our research project. During this research, Arianna and I will learn techniques in protein mutagenesis as well as how to characterize hydrophobic and electrostatic protein-protein interactions. The methods we will be using in the future to answer our research questions include: expressing, purifying, manipulating and characterizing proteins; forming and refining silica mineralization assays, both in the absence and presence of proteins; completing scanning and transmission electron microscopy, atomic force microscopy and focus-ion beam milling; and finally, analyzing our complex datasets in order to accurately interpret the results.

This is my first time working in a research lab so each step along the way has been a new opportunity for me to learn. From the first day learning how to use the autoclave, to our last meeting when we successfully transformed plasmid containing the cg-LDH gene into a host organism, I have learned a wealth of new lab skills. Exposing myself to new academic experiences and growing as a student as well as a team member are the key things I would like to achieve from this project. Through our research, I hope to expand my knowledge of proteins, enzymes, and the amino acids which are the building blocks behind them.