Inserting phosphatase genes into an inducible plasmid vector for Lactococcus lactis farnesol production
Faculty Mentor
Luis Matos
Document Type
Poster
Start Date
10-5-2023 11:15 AM
End Date
10-5-2023 1:00 PM
Location
PUB NCR
Department
Biology
Abstract
Farnesol (FOH), a molecule shown to reduce inflammation in the multiple sclerosis (MS) mouse model, has a precursor molecule called farnesyl pyrophosphate (FPP), that is produced in the mevalonate pathway (MVA). Certain phosphatases can catalyze the conversion of FPP to FOH. This metabolic pathway has been successfully engineered in E. coli. FOH production has not yet been studied in L. lactis, but the bacterium already contains the MVA and has application as an engineered probiotic far beyond the scope of E. coli. We hypothesize that L. lactis can be engineered to produce FOH by inserting genes for phosphatases (Pgpb, Phos15739, and YbjG) into a vector, and transforming it into the bacterium such that the bacterium will be able to convert FPP to FOH. Molecular cloning techniques are used to modify the vector plasmid and add the proper genes. Once these genes are added, the plasmid is transformed into L. lactis. Insertion is confirmed with selective media using antibiotics. The vectors pNZ7021 and pNZ8150 (Boca Scientific) will be used to introduce the phosphatase gene into L. lactis. As an additional selection measure, we are first introducing a second antibiotic resistance gene (erythromycin resistance) into these vectors. Subsequently, we will introduce a phosphatase gene from E. coli (EcoPhos) that catalyzes FPP to FOH. Successful integration of genes into respective vectors will be confirmed by PCR.At present, we have successfully ligated the ERM gene into pNZ7021. We are continuing work toward introducing the phosphatase gene into pNZ7021. Once these vectors have been successfully constructed, we will move to transform a ligation of PnZ+eRM+EcoPhos into L. lactis MG1363 cells to determine enzyme production efficiency and FOH production.
Recommended Citation
Betz, Elizabeth, "Inserting phosphatase genes into an inducible plasmid vector for Lactococcus lactis farnesol production" (2023). 2023 Symposium. 15.
https://dc.ewu.edu/srcw_2023/res_2023/p2_2023/15
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Inserting phosphatase genes into an inducible plasmid vector for Lactococcus lactis farnesol production
PUB NCR
Farnesol (FOH), a molecule shown to reduce inflammation in the multiple sclerosis (MS) mouse model, has a precursor molecule called farnesyl pyrophosphate (FPP), that is produced in the mevalonate pathway (MVA). Certain phosphatases can catalyze the conversion of FPP to FOH. This metabolic pathway has been successfully engineered in E. coli. FOH production has not yet been studied in L. lactis, but the bacterium already contains the MVA and has application as an engineered probiotic far beyond the scope of E. coli. We hypothesize that L. lactis can be engineered to produce FOH by inserting genes for phosphatases (Pgpb, Phos15739, and YbjG) into a vector, and transforming it into the bacterium such that the bacterium will be able to convert FPP to FOH. Molecular cloning techniques are used to modify the vector plasmid and add the proper genes. Once these genes are added, the plasmid is transformed into L. lactis. Insertion is confirmed with selective media using antibiotics. The vectors pNZ7021 and pNZ8150 (Boca Scientific) will be used to introduce the phosphatase gene into L. lactis. As an additional selection measure, we are first introducing a second antibiotic resistance gene (erythromycin resistance) into these vectors. Subsequently, we will introduce a phosphatase gene from E. coli (EcoPhos) that catalyzes FPP to FOH. Successful integration of genes into respective vectors will be confirmed by PCR.At present, we have successfully ligated the ERM gene into pNZ7021. We are continuing work toward introducing the phosphatase gene into pNZ7021. Once these vectors have been successfully constructed, we will move to transform a ligation of PnZ+eRM+EcoPhos into L. lactis MG1363 cells to determine enzyme production efficiency and FOH production.
