This post is written by UT undergraduate researchers Zachary Martinez and Andrew Ly
The University of Texas at Austin is known for many things: from being a powerhouse in Division 1 sports, to leading the world in innovation and cutting-edge research. However, there is one historic fact that many Longhorns do not know, and that is the success of the UT Austin iGEM team. For the past six years at the International Genetically Engineered Machine (iGEM) conference, UT Austin has earned a gold medal each year, an honor bestowed only to teams fulfilling the highest and strictest research requirements. This annual synthetic biology conference takes place in Boston, where over 300 teams from universities around the world present their research. This year, the UT Austin team consisted of a wide range of students, from underclassmen that have just started doing research, to more seasoned upperclassmen that have participated in iGEM previously. The 2017 project focused on engineering an effective GABA-producing probiotic.
The indigenous gut flora of humans possesses the ability to synthesize neurotransmitters, such as GABA, that are hypothesized to influence behavioral, cognitive, and emotional processes of the body via the gut-brain axis. The microbiome-gut-brain axis is a bi-directional communication system in which the microbiome of the gut affects the central nervous system, and vice-versa. Using this information along with our background in microbiology, molecular biology, and synthetic biology, we set out to engineer this microbiome as a way to potentially treat mental illnesses.
Gamma-Aminobutyric acid, or GABA, is the chief inhibitory neurotransmitter in the body and is responsible for reducing neuronal signaling in the central nervous system. Medications, such as alprazolam and diazepam, that increase GABA signaling are typically used for treating anxiety disorders. However, such drugs can lead to a physical dependence, and if given to children, a “pill-popping” habit. Due to these reasons, we began researching potential probiotics that we could study and engineer in order to produce GABA. We ended up picking Lactobacillus plantarum, which is not only indigenous to the human gut, but also expresses GABA in small amounts by converting glutamate to GABA via a glutamate decarboxylase enzyme encoded by the gadB gene. Our goal was to engineer this microbe to produce high levels of GABA and implement it into fermentable foods (such as kombucha, kimchi, or yogurt), which could then be ingested as an alternative form of medicine for patients suffering from anxiety.
In order to engineer our probiotic to produce high levels of GABA in the human gut, we first wanted to assemble a plasmid in which the gadB gene was overexpressed. To accomplish this, we employed a cloning technique called Golden Gate Assembly, which utilizes type IIS restriction enzymes that cut adjacent to the recognition sites. This allows for the scarless and simultaneous interchanging of different DNA parts, such as origins of replication, antibiotic resistance cassettes, coding sequences, and promoters, all while maintaining directionality in a single reaction. As such, we chose this assembly method due to its ability to rapidly create functional plasmid prototypes that would allow us to interchange parts quickly as we begin experimenting with L. plantarum. After successfully assembling our intended gadB overexpression plasmid using Golden Gate Assembly, we would then introduce it into our probiotic.
While trying to overexpress GABA, we observed various mutational inactivations of our gadB gene. Given that glutamate is an important substrate in biosynthesis and that GABA production requires the conversion of glutamate into GABA, we hypothesized that the functionally active form of gadB was ultimately toxic to cells. As a result, cells containing a mutated gadB gene were more evolutionarily fit and thus selected for. This explains why we were only able to obtain cells with the mutated gadB gene. We then constructed plasmids with either lower copy numbers and/or inducible promoters that would downregulate or control the expression of the gadB gene. However, we still found mutations within the gadB gene. Some possible solutions to address this issue are to utilize an inducible promoter with tighter regulation in our plasmid assembly, perform DNA transformations with a strain with a lower mutation rate, or even simply growing the bacteria in media supplemented with high levels of glutamate. Our future directions include developing a quorum sensing system in our engineered probiotic for controlled GABA production and potentially introducing our probiotic into the microbial ecosystem of the fermented beverage, kombucha, which was the main focus of our iGEM project last year. This year’s project, much like our 2015 iGEM project regarding evolutionary stability, has highlighted the importance of creating evolutionarily stable genetic circuits with low metabolic burdens: a problem synthetic biology has long had.
Overall, the iGEM conference was an invaluable experience where we were able to meet and network with numerous people from around the world, ranging from China to Ghana. We spoke to researchers who were looking into creating more robust genetic systems in a wide array of bacteria, something we have had an interest in for several years. Additionally, students from Vilnius, Lithuania discussed how they were able to use multiple plasmids within a single bacterium while controlling the copy number and maintaining this entire set of plasmids (five in total!) over multiple generations. As we prepare for next year’s iGEM competition, we hope to take what we have learned from this year’s experience and apply it to our 2018 research project.