Engineering Overview

This year, our team incorporated the engineering design process to identify aptamers and design/construct a plasmid to express alpha-amylase. Within our design process, we also used aptamers to identify a relative quantity of alpha-amylase protein. From this, we learned about some of the challenges of performing restriction digests, as well as what we would have done differently.

Design

As high school students, all of us are subject to high amounts of stress dealing with the pressure of preparing for difficult exams, college preparation, and lengthy homework assignments as well as the difficulties caused by the pandemic. Methods of detecting stress exist but most are either extremely time-inefficient and/or expensive. This intersection between stress and patient care inspired us to find a brand new method of detecting stress that can be easily accessible and administered.

We proposed saliva as the biological medium to detect stress because it is non-invasive, as there is no need to extract blood or cause unnecessary harm to the body. Our next step was to choose biomarkers in the saliva that would act as the best indicator of stress. Cortisol and alpha-amylase were easy to choose because they have been widely acknowledged among the scientific community as salivary stress biomarkers, with cortisol even being coined “the gold standard marker of stress.”

In our research for selecting salivary biomarkers, we came across an article published by Dr. Conrad Wiegand, “Stress-associated changes in salivary microRNAs can be detected in response to the Trier Social Stress Test: An exploratory study” (Wiegand 2018), which found significant associations with stress in three microRNAs: miR-20b, miR-21, and miR-26b. Our lab advisors Steven Reddy and Özge Begli suggested we use aptamers as a method for detecting cortisol and alpha-amylases, a relatively newer and less researched detection method that is designed to bind to target molecules. These aptamers emit fluorescence as an indication of the presence of those target molecules.

Build

Our team engineered alpha-amylase plasmid DNA containing two components: the human alpha-amylase insert gene (gene of interest, GOI) and the excised pET-28a(+)-FGF2-G3 plasmid (linearized backbone).

We bought the human alpha-amylase insert gene and used PCR (polymerase chain reaction) to acquire copies of the human alpha-amylase insert gene to use for the construction of the recombinant plasmid. For the linearized backbone, we first grew an overnight bacterial culture containing colonies with the plasmid PET-28a(+) using the Addgene protocol on inoculating a liquid bacterial culture. Once we got enough bacterial colonies as a result of the overnight culture, we isolated the plasmid PET-28a(+) in the colonies using the directions from the QIAprep Spin Miniprep Kit.

Afterwards, we used the enzymes XbaI and XhoI on the plasmid PET-28a(+) that we obtained through a process known as a restriction digest, in which the two restriction enzymes excised (cut out) a piece of the plasmid backbone that we would eventually replace with the human alpha-amylase insert gene. We used gel extraction to isolate the broken fragments of the plasmid backbone. Then we put together the human alpha-amylase insert gene with the linearized backbone to form the recombinant plasmid using a Gibson assembly kit.

After cloning and isolating our recombinant plasmids made from the GOI and linearized backbone through the use of DH5a E. coli cells, we transformed them into BL21 E. coli cells. BL21 cells are a commonly used bacterial strain for the production of recombinant proteins, including alpha-amylase. After transformation, the BL21 bacterial culture secreted alpha-amylase proteins.

Test

After creating the artificial alpha-amylase, we wanted to test the aptamer detection method using saliva models containing differing concentrations of alpha-amylase. Initially we planned on using fluorescence-based aptamers that would first bind to target proteins in an assay and emit a certain level of light intensity depending on the concentration of that target protein. However, as we continued our experiments, it became apparent to us that these types of aptamers were not suitable for detecting alpha-amylase because of the requirements of needing a fluorescent plate reader, leading us to look at aptamers that did not emit fluorescence. These aptamers could only be used to assess the concentration of the aptamer and protein complex by running the assay in a polyacrylamide gel electrophoresis, and depending on the distance the molecules travel, we could determine how much of the target protein was in the solution/sample.

Learn

During our lab work, one of the problems that we encountered was performing the restriction digest to insert the PCR product and attempting to make our Alpha-Amylase with an unpopular plasmid (pET-28A), running into situations where it simply would not work. One of the major downsides of this was the lack of resources, such as prior research using the same plasmid, to turn to when we ran into such issues due to our plasmid’s unpopularity. One thing we could have done was to work with a more reliable and historically tested plasmid instead.