Revision as of 05:58, 27 October 2020

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.

Sequence and Features