Antibiotic Resistance Gene Free Integration of a High GABA-Producing Gene Cassette into the Lactococcus lactis Chromosome Using a CRISPR-Cas/Transposon System
Faculty Mentor
Andrea Castillo
Presentation Type
Poster
Start Date
4-14-2026 2:00 PM
End Date
4-14-2026 4:00 PM
Location
PUB NCR
Primary Discipline of Presentation
Biology
Abstract
As links between the gut microbiota and human health emerge, so have efforts to develop engineered probiotics as therapeutics. The gut microbiota consists of viruses, bacteria, and fungi that produce chemicals, such as g-aminobutyric acid (GABA), that impact human health. GABA is the primary inhibitory neurotransmitter, controlling synapses and brain development; it also plays a role in mental health and inflammatory-based diseases. Efforts have been made to identify or engineer high-GABA-producing bacteria as possible therapeutics. Previously, our lab engineered a lactic acid bacterium (LAB), Lactococcus lactis to produce high GABA levels. This strain, GAD-L. lactis, has the high GABA synthesizing gene cassette, GABAcass, on a plasmid, that also carries an antibiotic resistance (antR) gene. The presence of the antR gene in this strain makes the engineered probiotic unsuitable for development as a therapeutic. My project is to overcome this impediment by integrating GABAcass, minus the antR gene, into the L. lactis chromosome using a two-plasmid (pDonor-mod and pINT) CRISPR system. This system will allow integration of GABAcass from pDonor-mod to the lacZ chromosomal locus of L. lactis, using machinery encoded in the pINT plasmid. Currently, we are working to clone GABAcass into pDonor-mod and assemble pINT from three commercially synthesized DNA fragments. Once assembled, the plasmids will be transformed into L. lactis and tested for high GABA synthesis using an Enzyme Linked Immunosorbent Assay. In experiments beyond the scope of this project, high GABA producing L. lactis will be tested in animal models of disease as a potential therapeutic.
Recommended Citation
Phillips, Christoff, "Antibiotic Resistance Gene Free Integration of a High GABA-Producing Gene Cassette into the Lactococcus lactis Chromosome Using a CRISPR-Cas/Transposon System" (2026). 2026 Symposium. 25.
https://dc.ewu.edu/srcw_2026/ps_2026/p3_2026/25
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Antibiotic Resistance Gene Free Integration of a High GABA-Producing Gene Cassette into the Lactococcus lactis Chromosome Using a CRISPR-Cas/Transposon System
PUB NCR
As links between the gut microbiota and human health emerge, so have efforts to develop engineered probiotics as therapeutics. The gut microbiota consists of viruses, bacteria, and fungi that produce chemicals, such as g-aminobutyric acid (GABA), that impact human health. GABA is the primary inhibitory neurotransmitter, controlling synapses and brain development; it also plays a role in mental health and inflammatory-based diseases. Efforts have been made to identify or engineer high-GABA-producing bacteria as possible therapeutics. Previously, our lab engineered a lactic acid bacterium (LAB), Lactococcus lactis to produce high GABA levels. This strain, GAD-L. lactis, has the high GABA synthesizing gene cassette, GABAcass, on a plasmid, that also carries an antibiotic resistance (antR) gene. The presence of the antR gene in this strain makes the engineered probiotic unsuitable for development as a therapeutic. My project is to overcome this impediment by integrating GABAcass, minus the antR gene, into the L. lactis chromosome using a two-plasmid (pDonor-mod and pINT) CRISPR system. This system will allow integration of GABAcass from pDonor-mod to the lacZ chromosomal locus of L. lactis, using machinery encoded in the pINT plasmid. Currently, we are working to clone GABAcass into pDonor-mod and assemble pINT from three commercially synthesized DNA fragments. Once assembled, the plasmids will be transformed into L. lactis and tested for high GABA synthesis using an Enzyme Linked Immunosorbent Assay. In experiments beyond the scope of this project, high GABA producing L. lactis will be tested in animal models of disease as a potential therapeutic.
