At the beginning of this program, I remembering feeling nervous about my lack of experience in a lab setting. I was worried I wouldn’t understand the complex planning and decision making that occurred in research, and that I would get lost in all the information. Over time and after some trial and error, I’m glad to say I have been proven wrong.
I was able to learn so many different concepts through the guidance of my mentor and hands-on experience, allowing me to apply these ideas in our project. It was definitely hard when first starting, but I quickly adapted and gained a passion for our research and its applications. This experience has given me a newfound confidence in participating in scientific research and discussion. It also showed me how much I enjoy research and introduced me to topics I found fascinating, and hopefully in the future I will be able to continue my work in the Chilkoti lab.
This summer has been one of the most fun and fulfilling I have ever experienced, and I look forward to my future work in research!
Mentors: Anya Varanko, Ashutosh Chilkoti, PhD.
Department of Biomedical Engineering
Elastin-like polypeptides (ELPs) are proteins based off of tropoelastin, a subcomponent of elastin, that are able to change solubility under different temperatures. By combining these proteins with the affibodies ZEGFR and ZHER2, the proliferation of cancer can be limited much more effectively than bispecific antibodies. ELPs were combined with affibodies in E. coli plasmids to create different protein constructs, purifying the proteins using heat cycling. SK-BR-3 cancer cells positive for both EGFR and HER2 were then treated with the previous proteins to create a proliferation assay. Each construct maintained the same amount of proliferation as the control at all concentrations, resulting in no color change for the assay. This suggests that the constructs had no effect on the growth of SK-BR-3, and both affibodies were unable to block the receptors. The potential of ELPs and bispecifics still require further investigation, as it may be possible ELPs inhibit the binding of the affibodies, that ZEGFR and ZHER2 are incompatible with ELPs, or other outside factors. The use of other bispecifics, as well as maintaining temperature while performing the assay, will be explored as research in ELPs continue.
I thoroughly enjoyed hearing about the different projects everyone was working on this summer, and was pleasantly surprised to see a large variety in the topics. One such presentation that interested me was Ben’s talk about cell migration. He explained how cells can use force on actin filaments to communicate to each other, effectively causing the cells to move together. Specifically, he is looking into how vinculin can play a part in cell migration, and if it can be used to control cell movement in the future. This topic specifically fascinates me due to its similarity with my project, in which both of us look at unique characteristics in certain materials and try to optimize them for medical purposes.
Ben explained that cell migration could potentially be used for wound healing and tissue regeneration, which is actually similar to applications of ELP’s. ELP’s can also be used in damaged joints or tissue and become a solidified deposit to protect certain areas. It was exciting to learn of other possibilities in tissue engineering that share a lot of similarities with my work! Ben’s engaging and detailed talk makes me look forward to the future work his lab and others will do in the field of tissue engineering and biomaterials, as well as other possible solutions that will arise as research continues.
In the Chilkoti lab, we work to create different proteins that could potentially improve drug delivery. To do this, bacteria must be constantly transformed, grown, and lysed. Each step takes hours, and depends on whether or not the previous step was successful. Because of this unpredictability, workdays can only be planned one or two days before. While each day may seem similar, every step is important for making a functional protein we can store and use for future purposes.
Some days we start at the beginning, with different pieces of plasmids we have to recombine. After cutting the plasmids using certain types of enzymes, we have a new piece of DNA that can be transformed into bacteria using a heat shock. These bacteria now have new plasmids that contain our desired protein sequence of ELP, and we allow them to create the protein as usual overnight or after a day. This step can take many hours, but necessary to allow for a greater yield of protein.
After a large amount of bacteria have grown, the protein must be extracted by lysing the cells. This erupts the membrane and allows the protein to be released into the surrounding solution. Afterwards, the desired protein must be separated and purified from other contaminants, which can be done by utilizing the ELP’s change in solubility under different temperatures. By switching between hot and cold temperatures and centrifuging the solution, the ELP can be isolated from other proteins that are always either soluble or insoluble. Through these cycles, the ELP becomes purified and ready to use for further testing.
It is important to note that these steps can be unsuccessful, and so a large amount of time is also devoted to checking our work and making sure we can continue with the purification. Without running gels to check DNA or protein size, we can’t be sure if the results we find are accurate. Although frustrating at times, the payoff of finding our sample to be purely our desired protein can make the days-long process worth it.
My PI, Dr. Chilkoti, was introduced to research and science in general a bit differently than others: through necessity. He grew up in India, and after high school he didn’t have many options for higher education. He entered college and began studying engineering, but he found himself lacking passion or interest in his studies. He also worked towards an MBA, which he thought was even more disinteresting, and found business to be “bogus” to him. In the end, he decided he wanted to explore more options, but most importantly outside of India.
Dr. Chilkoti set his sights on the US as the best place to find better opportunities, but he was struggling to find the funds for such a move. He only had enough money for a plane ticket, and so he began searching for other ways to afford living in the US. He was able to find and apply for a PhD program that would pay for his living expenses, and he was accepted to work on his own project. Because of the program, he was able to move out of India and work on his own terms, which he preferred much more over working for a business. He enjoyed exploring hypotheses and asking difficult questions, and in the end began to work towards a postdoc.
Around this time, he changed his focus to molecular biology, and that was when he believed he found his passion. He enjoyed his work and continued to studying molecular biology at his job at Duke, where he is currently working on the study of proteins for drug delivery. His favorite part about his job is having the chance to discover new aspects of molecular biology and combine them with engineering to further improve the scientific field.
In the end, I asked Dr. Chilkoti if he had any advice for someone beginning their search for a career. Staying true to his story, he told me to “follow my passion,” and never try to choose a career based solely on money or stability. No matter what path you choose, Dr. Chilkoti suggests “if you are excited, you will do well.” Speaking with Dr. Chilkoti was a great experience, and I look forward to continue researching in his lab this summer.
The field of drug therapy and molecular engineering is constantly changing and exploring new options to improve efficiency. One main issue when creating new drugs is controlling how long it can stay in someone’s system before it is removed, as well as accuracy. Many research labs have begun looking into new polymers, especially ones that can be grown in bacterial cultures, to assist in the transportation of drugs. I am working this summer in the Chilkoti Lab to investigate a biopolymer that is being tested as a future drug delivery option.
My main project involves assisting my lab mentor, Anastasia Varanko, in growing and harvesting elastin-like polypeptides. Also known as ELPs, these polymers can be grown in bacteria and have a unique property of changing solubility based on temperature. When it is at lower temperatures, it becomes more soluble, and at higher temperatures it can become insoluble. This temperature threshold can be modified through molecular engineering, and allow scientists to control when it aggregates. It is then possible to keep the ELP at a lower temperature for first injecting the drug, and after entering the body it can become insoluble and extend its circulation in the blood. This leads to an overall more long-lasting and efficient drug. Other proteins can also be added to the ELPs, which may be utilized to transport proteins into the body. This includes receptor blockers that can inhibit or enhance different pathways.
My main focus will be collecting ELPs from bacteria and purifying them, as well as attaching different proteins to the ELPs using transformation on bacteria. I look forward to continuing my work in the lab throughout this summer and hopefully creating a stable protein that can be used for future drug applications.
When first entering the Chilkoti Lab, you’ll take notice of the giant flasks around the room filled with dark golden liquids and covered in foil. Like most other things in the lab, these flasks are one of the many tools used to grow and collect bacteria. The Chilkoti Lab focuses on working in the microscopic world as they try to collect ELPs, proteins that are soluble and insoluble under certain temperatures and can be created by bacteria. These are then studied to see how to best use ELPs to improve drug delivery and therapy as a whole. To collect these proteins, a variety of instruments and procedures must be employed.
I have not had much research experience in my past. I have never officially worked in a lab or carried out complex experiments, and so my knowledge on how a lab functions was minimal. Entering the Chilkoti Lab, I felt slightly nervous and intimidated by what I was about to begin working on. Thankfully, my mentor greeted me with plenty of advice and support as she began to teach me more about mini preps, creating petri dishes, and other essential skills. Over the summer, I hope to continue learning more about the bacterial and molecular world, as well as increase my knowledge of drug therapy. I also hope to gain more experience carrying out common protocols and remembering important information that is relevant to the lab. Finally, I hope that I will be able to gain some form of independence over the summer and allow myself to get used to the usual responsibilities of the lab on my own.
I expect this summer to be difficult at first, especially when having to keep track of all the steps in a protocol or learning where everything is located around the lab. Yet, I also expect I will learn first-hand how to conduct scientific research and the challenges that come along with it.