Team:Mingdao/Composite Part
Mingdao
This year, we designed, constructed and demonstrated a composite part for genetically engineering probiotics to combat oral etiological pathogens. Such a 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.
Fig. 1. The schematic diagram of genetically engineering concept in our study.
For the detail of gene cloning and part construction, please go to check our Part Collection
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.
Fig. 2. Promoters in this study. The GFP was detected at Ex/Em = 483/513. (A) Basic part: ldhp/pSB1C3 (BBa_K3376000), Composite part: ldhp-GFP-Tr/pSB1C3 (BBa_K3376002), (B) Basic part: tpxp/pSB1C3 (BBa_K3376003), Composite part: tpxp-GFP-Tr/pSB1C3 (BBa_K3376004), (C) GFP was expressed at different levels with the indicated promoters. Inset plot: GFP fluorescence under a blue LED box.
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.
Fig. 3. AQP facilitates H2O2 permeation. (A) Basic par: AQP/pSB1C3 (BBa_K3376005) and Composite part: ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 (BBa_K3376007), (B) GFP expression level was increased with AQP in the presence of H2O2 compared to the control without AQP.
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.
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.
Fig. 4. The promoter activity of tpxp was increased dose-dependently with H2O2 concentration in the presence of AQP.
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).
Fig. 5. SpxB produces H2O2. The H2O2 was measured with a Fluorimetric Hydrogen Peroxide Assay Kit (SIGMA-ALDRICH). (A) Basic part: SpxB/pSB1C3 (BBa_K3376008) and Composite part: ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa_K3376010), (B) The H2O2 was produced by the transformants expressing AQP and SpxB.
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.
Fig. 6. Growth rate of E. coli Nissle transformed with ldhp-AQP-RBS-SpxB-Tr/pSB1C3 was measured at OD600. The cells were cultured in LB broth supplemented with 1%, 5% or 10% glucose for 5 hr. The cell growth was severely inhibited compared to the vector-only control.
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.
In our experiment, 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.
In our experiment, 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. 7. Growth recovery and the function of KatG. (A) An existing BioBrick part of KatG-Tr/pSB1AK3 (BBa_S04059) (B) Growth rate of E. coli Nissle transformed with the indicated plasmid was measured at OD600. The cells were cultured in LB broth supplemented with 1%, 5% or 10% glucose for 5 hr.
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.
Fig. 8. Protein expression with Coomassie blue staining on 4 – 20% gradient SDS-PAGE. E. coli Nissle carrying the indicated plasmid vector was cultured in LB supplemented with 34 µg/ml of chloramphenicol. The lysates were harvested from overnight culture and 5 µg of them was subjected to SDS-PAGE.
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.
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.
Fig. 9. H2O2 production of E. coli Nissle transformed with the indicated plasmid were measured and normalized by the values of OD600. The cells were cultured in LB broth supplemented with 1%, 5% or 10% glucose for 5 hr.
Taken together, based on the results of experiments of H2O2 transportation, H2O2-regulated promoter activity, the growth recovery by catalase, and H2O2 production assay, as well as protein expression analysis, all the data demonstrated the functionality of our composite BioBrick device.
Taken together, based on the results of experiments of H2O2 transportation, H2O2-regulated promoter activity, the growth recovery by catalase, and H2O2 production assay, as well as protein expression analysis, all the data demonstrated the functionality of our composite BioBrick device.
PROOF-OF-CONCEPT
Antagonism
In the natural environment, S. sanguinis and S. mutans are antagonists which can inhibit the growth of each other. As a result, they are usually used as a model to probe the competition between different species that occupy the same ecological niche. We conducted the experiments of antagonism test between S. mutans and S. sanguinis, S. mutans and WT E. coli Nissle (EcN), S. mutans and GM EcN. The GM EcN has a strong ability to inhibit the growth of S. mutans as shown in the cell shape and size on the agar plates, and even better than S. sanguinis. There’s no antagonistic effect between S. mutans and WT EcN control. The results significantly indicated that the H2O2 produced by the device in GM EcN may be capable of efficiently eliminating S. mutans.
Fig. 10. Antagonism test between S. mutans and S. sanguinis (A), S. mutans and WT EcN (B), S. mutans and GM EcN (C). 8 µL of the overnight cultures adjusted to an OD600 of ~0.5 were inoculated on LB agar plates beside each other. The data showed two repeats observed after 24-hr culture.
Prototype
The handmade lozenge we created is a sweet candy made of 5% sugar alcohols (i.e, xylitol and erythritol) and weighs 1 gram. It contains approximately 3 x 108 CFU (colony forming unit) of EcN (E. coli Nissle) per tablet. To determine the protein expression in our prototype, we made three kinds of lozenges containing GM EcN, which carries the empty vector, the GFP expression device and the AQP-SpxB-KatG expression device. When exposed to blue LED light, EcN with GFP glowed brightly compared to the controls, indicating that GM EcN can express proteins even made into a candy. Moreover, to test the functionality of GM EcN with AQP-SpxB-KatG, we examined the production of H2O2 in the lozenges. The saliva production in the mouth will reach to 3 – 5 ml per minute during eating, chewing or other stimulating activities. Therefore, we dissolved the lozenge in the 3 ml of ddH2O for 5 min and measured H2O2 concentration. GM EcN with AQP-SpxB-KatG can produce 0.58 mM per OD600 compared to the background level (0.21 mM/OD600) by the vector-only control.
Fig. 11. The handmade lozenges contain GM EcN with the empty vector as control, GFP expression device, or AQP-SpkB-KatG expression device. (A) GFP was observed in a blue LED box. (B) The lozenges were dissolved in the ddH2O and tested for H2O2 production.
Taken together, the candy as a prototype we made with GM EcN not only carries live probiotics expressing proteins, but also produces H2O2, implying an effective product against S. mutans for maintenance of oral health.
Taken together, the candy as a prototype we made with GM EcN not only carries live probiotics expressing proteins, but also produces H2O2, implying an effective product against S. mutans for maintenance of oral health.