Lauren Binder, Sara Matsumoto, Natalia Trounce, Shawn Vinogradsky
BioBuilderClub, Palo Alto High School, Palo Alto, California, USA, and Carlmont High School, Belmont, California, USA
Reviewed on 2 May 2020; Accepted on 22 June 2020; Published on 26 October 2020
With help from the 2020 BioTreks Production Team.
Gluten is the common name for a group of complex proteins found in grains such as wheat, barley, and rye. There are four conditions associated with negative reactions caused by gluten consumption, which affect 5-10% of the global population: celiac disease, wheat allergies, gluten sensitivity, and gluten intolerance. In our experiment, we used Escherichia coli as a microbial host to produce the PEP 2RA3 microbial enzyme, a bacterial isolate that has been cloned and expressed in E. coli. It breaks down gliadins, one of two main protein groups in gluten.
These enzymes will be used in a pill (similar behaviors to the common lactase pill) with the purpose of helping affected individuals safely ingest foods containing gluten, in a more effective manner. For patients with celiac disease, our pill treatment may not be as effective as previously presumed, due to the fact that even the smallest particles of gluten can set off an immunogenic response. This reaction can damage villi lining the small intestine as there is a certain threshold for the amount of gluten that can safely be in the system of a patient with celiac disease. If gluten content is above this amount of 10-50 mg (consumed per day) then the patient is at risk of an autoimmune response that can damage the lining of the small intestine (villi) or cause cytokine storms. For people with other conditions (wheat allergies, gluten sensitivity, and gluten intolerance), however, the effects of gluten have the potential to be greatly reduced, even if there are small particles of gluten remaining after degradation. This pill would allow users to find enjoyment in the taste, smell, and texture of gluten-containing foods, while avoiding the majority of debilitating symptoms that they would otherwise face.
Keywords: gluten, gluten degradation, gliadin, glutenin, celiac disease
Authors are listed in alphabetical order. Vandon Duong and David Lips mentored the group. Please direct all correspondence to firstname.lastname@example.org.
There are four conditions associated with negative reactions caused by gluten consumption, that affect 5-10% of the global population: celiac disease, wheat allergies, gluten sensitivity, and gluten intolerance (Brazier 2020). In this project, we used Escherichia coli as a microbial host to produce the PEP 2RA3 microbial enzyme (Figure 1), which breaks down gliadins, one of two main protein groups in gluten (Amador, Arévalo-Rodríguez, Durán, et al. 2019). The E. coli will be used in a pill which helps individuals with gluten sensitivities safely ingest foods containing gluten. Even if small particles of gluten remain after degradation, this idea may be beneficial because it allows people to enjoy gluten-containing foods, without the consequences they would otherwise face.
|Figure 1. A basic plasmid containing the enzyme, promoter, RBS, and terminator. Parts may be added as needed to purify the enzyme (International Genetically Engineered Machine 2020).|
We created a basic DNA construct (See Figure 2), consisting of a promoter (start) sequence, a ribosome binding site (RBS), the enzyme coding sequence (CDS) for the PEP 2RA3, and a T7 terminator sequence (Figure 3). The terminator and promoter were both chosen from the iGEM parts registry (International Genetically Engineered Machine 2020). To convert the construct into a plasmid (Figure 1), we modified the ends of the restriction sites and put them together with ligase. To produce the enzyme, we would transform this plasmid into E. coli, and place the E. coli inside a pill to be taken orally. However, the enzyme needs to be purified before it is placed in the pill for consumption, and tested in conditions that mimic the human intestine. For this design, IPTG would not need to be present in order to activate transcription of the lac operon and express the enzyme as we are using an RBS sequence from the Anderson promoter library (Anderson 2006). We also need to test how the body will react to this enzyme, and how long it will take for it to destroy the protein for not having human similarity and glycosylation pattern. One way to test our design would be to see if E. coli can efficiently secrete the enzyme in the proper conditions and whether the enzyme degrades gluten in the typical range of pH and temperature of the human body, which might be measured with petri plates containing gliadins. We could also purify the enzyme from E. coli without secretion, and test the efficiency in-vitro without E. coli, just the enzyme.
|Figure 2. A simple construct for the DNA. It consists of a promoter region, the ribosome binding sequence, the PEP 2RA3 enzyme, and a T7 terminator (International Genetically Engineered Machine 2020).|
|Figure 3. The sequence of DNA with a promoter, RBS, terminator, and the full PEP 2RA3 enzyme sequence (International Genetically Engineered Machine 2020).|
The PEP 2RA3 enzyme is purified and expressed in E. coli. The E. coli is placed in a pill to be ingested, upon which the enzymes break down gliadin proteins in the gut. The gluten content after degradation needs to be below a specific amount (about 10-50 mg, consumed per day) in order for the body’s reaction to be reduced or relieved.
The key components of this design include the engineered E. Coli, the PEP 2RA3 enzyme, which will degrade gliadin proteins, and the pill which will transport the modified enzyme into the intestine to act upon the consumed gluten. It is uncertain how long it will take the body to destroy the ingested protein, as it does not have the human similarity or glycosylation pattern, and this needs to be tested- however, it is a reasonable conjecture to say that it should be enough time for much of the gluten in the system to degrade thanks to the embedded PEP 2RA3 enzyme. In order to ensure high gene expression of the enzyme and thus a greater amount of gluten degraded, we chose a strong constitutive promoter from the Anderson promoter library, located in the iGEM repository, as well as an RBS sequence from the Anderson RBS library to make sure translation is initiated properly.
The plasmid containing the enzyme and necessary parts such as the promoter, RBS and terminator (International Genetically Engineered Machine 2020) will be put together in a pill and ingested. Upon arrival to the intestine, the enzyme will be activated and begin to digest gluten found in the gut (Amador, Arévalo-Rodríguez, Durán, et al. 2019).
This is a design brief, so we are not yet certain of the specific methods used to test this design. However, we are certain that testing is a crucial aspect of this idea and many clinical trials will need to be undergone in order to make sure that this pill is safe for human consumption.
Additional testing will be needed to perceive the effectiveness of this enzyme in degrading gluten, and to be absolutely certain that this pill is ready and safe for consumption by humans.
We would like to thank Vandon Duong and David Lips, our mentors, teachers, and providers of knowledge. We recognize the BioBuilderClub organization for providing us with the opportunity and making it possible to design this biotechnology. Thank you to our team members who helped make all of this possible with all their hard work and time, and thank you to BioTreks for giving us the possibility to publish this article.
This project was accomplished through participation in the BioBuilderClub, an after-school program organized by BioBuilder Educational Foundation. BioBuilderClub engages high school teams around the world to combine engineering approaches and scientific know-how to design/build/test their own project ideas using synthetic biology.
Amador MDLM, Arévalo-Rodríguez M, Durán EM, Reyes JCM and Martín CS. A new microbial gluten-degrading prolyl endopeptidase: potential application in celiac disease to reduce gluten immunogenic peptides. Plos One. 2019 Jun 27 [cited 2020 Apr]];14(6). Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0218346
Brazier Y. What is gluten intolerance: symptoms and intolerance vs. allergy [Internet]. Medical News Today. MediLexicon International; 2020 Jan 28 [cited 2020 May 30]. Available from: https://www.medicalnewstoday.com/articles/312898#intolerance-allergy-celiac
International Genetically Engineered Machine. iGem 2020: an exceptional year [Internet]. Boston (MA): International Genetically Engineered Machine, Registry of Standard Biological Parts; c2020 [cited 2020May30]. Available from: http://parts.igem.org/Main_Page
Anderson CJ. Promoters/Catalog/Anderson [Internet]. 2006 [cited 2020]. Available from: http://parts.igem.org/Promoters/Catalog/Anderson