ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3

The device contains pyruvate oxidase (SpxB) gene from S. sanguinis to generate H2O2, aquaporin (AQP) gene from S. cristatus to facilitate H2O2 transport, and catalase (KatG) gene from E. coli to revive from oxidative stress by decomposing H2O2 to oxygen and water. The SpxB gene is driven under the constitutive and strong promoter of lactate dehydrogenase (ldhp) of S. mutans. The KatG gene is regulated under the promoter of thiol peroxidase (tpxp) of S. mutans, which is induced by H2O2. Therefore, catalase is expressed in the presence of H2O2 and detoxify the H2O2. Furthermore, the endogenous gene of lactate dehydrogenase is broken to eliminate the production of lactic acid.

THE PROMOTERS

First of all, two kinds of promoters from S. mutans were demonstrated. Lactate dehydrogenase promoter (ldhp) is a strong and constitutive promoter, and thiol peroxidase promoter (tpxp) is induced by the presence of H2O2 in the response to an environmental stress. The gene expression in E. coli Nissle was measured with GFP reporter by the fluorescence intensity at Ex/Em = 483/513 nm. The data showed a strong activity of ldhp and a limited expression of tpxp.

TRANSPORTER AND THE REGULATED PROMOTER

H2O2 permeability across the cell membrane is limited. The aquaporin (AQP) from Streptococcus cristatus facilitates bidirectional transmembrane H2O2 transport. When E. coli Nissle expressing AQP, GFP intensity was enhanced significantly in the presence of AQP and H2O2, indicating AQP functions as H2O2 transporter. The observation is consistent with the study by Huichun Tong, et al.

Furthermore, the GFP intensity was enhanced in response to the increasing concentrations of H2O2 in a dose-dependent manner, suggesting the promoter activity of tpxp was regulated by H2O2.

TOXIN GENERATOR

Pyruvate oxidase (SpxB) of S. sanguinis catalyzes the reaction between pyruvate and oxygen to generate acetyl phosphate and H2O2. It plays a role in antagonistic relationship against S. mutans. The E. coli Nissle carrying SpxB gene is capable of producing 2.15 mM and 1.84 mM of H2O2 per OD600 when grown in 5% and 10% glucose, respectively, compared to the background level of H2O2 production (i.e., 0.33 mM)

TOXIN DETOXIFIER

Catalase (KatG) catalyzes the H2O2 reduction to H2O and O2 and plays a role in E. coli to prevent DNA damage caused by an oxidative stress. The iGEM team Caltech in 2008 created the part of KatG (AHL-inducible KatG expression device, BBa_K137079) and demonstrated the function in minimum inhibitory concentration (MIC) assay, in which the cells were revived from H2O2 shock.

It expected that the bacterial growth was severely influenced in the presence of H2O2. Our data showed that the growth of E. coli Nissle cultured in different concentrations of glucose significantly retarded when expressing AQP and SpxB, further confirming the production of H2O2. In the presence of KatG, the growth rate was increased and comparable with the vector-only control, implied that KatG is capable of decomposing H2O2 and release cells from stress.

H2O2-PRODUCING DEVICE

The lysates of the transformed E. coli Nissle carrying the indicated gene were subjected to SDS-PAGE and Coomassie blue staining. When overexpression under the promoter of ldhp, KatG (80kDa) and AQP (17kDa) have sharp band on the PAGE. However, the predicted 65-kDa of SpxB was not detected, possibly because of the oxidative stress induced by the production of H2O2. In the presence of KatG, SpxB was shown up along with AQP, though expressed at a week level.

The E. coli Nissle carrying the device was cultured in LB broth supplemented with 1%, 5% or 10% glucose. The supernatants of 5-hr culture were subjected to H2O2 production assay. The amounts of H2O2 production were increased dose-dependently in response to the concentration of glucose.

Sequence and Features

Assembly Compatibility: