Team:Mingdao/Contribution
Mingdao
CHARACTERIZATION OF AN EXISTING BIOBRICK PART (BBa_S04059)
This year we’ve developed a H2O2-generating device in a probiotic E. coli Nissle. We need an antidote for the engineered cells to revive from the oxidative stress.
The basic part of BBa_S04059 contains KatG with a terminator from E. coli, which was created by iGEM team Caltech in 2008. KatG is bifunctional enzyme with catalase and peroxidase activity. It catalyzed the H2O2 reduction to H2O and O2, and plays a role in E. coli to prevent DNA damage caused by an oxidative stress.
In order to characterize the function of KatG, we assembled the KatG part into our functional device to create a composite part. In our experiment, we demonstrated the function of the catalase activity of KatG and protein expression in SDS-PAGE.
The basic part of BBa_S04059 contains KatG with a terminator from E. coli, which was created by iGEM team Caltech in 2008. KatG is bifunctional enzyme with catalase and peroxidase activity. It catalyzed the H2O2 reduction to H2O and O2, and plays a role in E. coli to prevent DNA damage caused by an oxidative stress.
In order to characterize the function of KatG, we assembled the KatG part into our functional device to create a composite part. In our experiment, we demonstrated the function of the catalase activity of KatG and protein expression in SDS-PAGE.
Fig. 1. The cell growth rate of E. coli Nissle with KatG at OD600 in various concentrations of glucose was comparable with the vector-only control, compared to the severe growth inhibition in E. coli without KatG. Our results strongly implied that KatG is capable of decomposing H2O2 and release cells from stress.
Fig. 2. 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.
SCREENSHOT OF THE PART REGISTRY DOCUMENTATION
