Team:Mingdao/Part Collection
Mingdao
This year, our goal is to create a probiotic strain to attack caries-associated pathogens in the mouth. For part collection, we considered the parts to be used in our device including a strong and constitutive promoter with an ability to highly express a gene, an inducible promoter regulated by the toxin, a gene which can produce toxin, and a gene that encodes antitoxin to protect cells from the toxin. Moreover, we built up a shuttle vector which can be used in gene cloning and has the ability to transform a broad range of host.
Fig. 1. The schematic diagram of categories, features, parts and functions collected in our study. The E in the open box refers to EcoRI site, X to XbaI, S to SpeI and P to PstI, which are standard restriction enzyme sites for BioBrick assembly.
Therefore, we have collected these parts in four categories with corresponding features and also demonstrated the functions of them in our experiments.
Table. 1. Th part name and its numbers used in our project.
CONSTITUTIVE PROMOTER
The ldhp (lactate dehydrogenase promoter) is a strong and constitutive promoter in S. mutans used and well-documented in many studies. Lactate dehydrogenase catalyzes the fermentation of pyruvate to lactic acid. It plays a pivotal role in the glucose metabolic pathway connecting glycolysis, and is recognized as an essential gene and expressed at high level in S. mutans.
The DNA fragment of ldhp with intrinsic RBS was amplified as an insert from gDNA of S. mutans by PCR and cloned onto pSB1C3 (BBa_K3376000). This basic part was further assembled with a reporter, GFP-Tr/pSB1C3 (BBa_K3376001), to generate ldhp-GFP-Tr/pSB1C3 (BBa_K3376002). All inserted sequences on the parts were confirmed by sequencing with VF2 and VR primes.
The ldhp (lactate dehydrogenase promoter) is a strong and constitutive promoter in S. mutans used and well-documented in many studies. Lactate dehydrogenase catalyzes the fermentation of pyruvate to lactic acid. It plays a pivotal role in the glucose metabolic pathway connecting glycolysis, and is recognized as an essential gene and expressed at high level in S. mutans.
The DNA fragment of ldhp with intrinsic RBS was amplified as an insert from gDNA of S. mutans by PCR and cloned onto pSB1C3 (BBa_K3376000). This basic part was further assembled with a reporter, GFP-Tr/pSB1C3 (BBa_K3376001), to generate ldhp-GFP-Tr/pSB1C3 (BBa_K3376002). All inserted sequences on the parts were confirmed by sequencing with VF2 and VR primes.
Fig. 2. The cloning of ldhp/pSB1C3 (BBa_K3376000) and ldhp-GFP-Tr/pSB1C3 (BBa_K3376002). (A) The ldhp was amplified as an insert by PCR with BioBrick prefix and suffix elements (216 bp) from gDNA of S. mutans. (B) Colony PCR check for ldhp-GFP-Tr/pSB1C3 with ldhp-forward primer and GFP-reverse primer (887 bp). (C) Restriction enzyme check for ldhp-GFP-Tr/pSB1C3 by EcoRI and PstI (2029 + 1024 bp).
The resulting ldhp-GFP-Tr/pSB1C3 (BBa_K3376002) was transformed into E. coli Nissle strain. The high expression of green fluorescent protein was observed under a blue led light.
Fig. 3. The GFP in the transformed E. coli carrying ldhp-GFP-Tr/pSB1C3 on the LB agar plate supplemented with 20 µg/ml of chloramphenicol was observed in the blue LED box.
TOXIN GENE
Abuse of antibiotics is a severe global health problem with the consequences of multidrug-resistant bacteria. S. sanguinis is a natural oral microbe species, which has antagonistic effect against etiological S. mutans through H2O2 production by pyruvate oxidase (SpxB).
In order to facilitate the H2O2 transport, we cloned an aquaporin (AQP) from S. cristatus based on the study by Huichun Tong, et al. The AQP is a transmembrane protein, which is capable of aiding in the bidirectional transportation of H2O2 in E. coli.
The SpxB (BBa_K3376008) and AQP (BBa_K3376005) gene sequences were both synthesized by IDT. The parts were assembled to pSB1C3 with ldhp (BBa_K3376000), an RBS (BBa_B0034) and a double terminator (BBa_B0015) to generate a composite part of ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa_K3376010). All inserted sequences on the parts were confirmed by sequencing with VF2 and VR primes.
Abuse of antibiotics is a severe global health problem with the consequences of multidrug-resistant bacteria. S. sanguinis is a natural oral microbe species, which has antagonistic effect against etiological S. mutans through H2O2 production by pyruvate oxidase (SpxB).
In order to facilitate the H2O2 transport, we cloned an aquaporin (AQP) from S. cristatus based on the study by Huichun Tong, et al. The AQP is a transmembrane protein, which is capable of aiding in the bidirectional transportation of H2O2 in E. coli.
The SpxB (BBa_K3376008) and AQP (BBa_K3376005) gene sequences were both synthesized by IDT. The parts were assembled to pSB1C3 with ldhp (BBa_K3376000), an RBS (BBa_B0034) and a double terminator (BBa_B0015) to generate a composite part of ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa_K3376010). All inserted sequences on the parts were confirmed by sequencing with VF2 and VR primes.
Fig. 4. The cloning of AQP/pSB1C3 (BBa_K3376005), SpxB/pSB1C3 (BBa_K3376008) and ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa_K3376010). (A) The AQP and SpxB were amplified as inserts by PCR with BioBrick prefix and suffix elements (554 bp and 1867 bp, respectively) from the synthesized gene on pUCIDT-Kan by IDT. (B) and (C) The plasmids were checked by colony PCR with ldhp-forward primer and SpxB-reverse primer (2473 bp) and by restriction enzymes of EcoRI and PstI (2651 + 2029 bp).
To determine the H2O2 production, we tested the transformed E. coli Nissle carrying ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (GM EcN) in the culture medium of LB supplemented with 5% or 10% glucose. The supernatants of overnight culture were subjected to H2O2 production measurement. The assay showed that 2.15 mM and 1.84 mM of H2O2 per OD600 were produced by GM EcN cultured in 5% and 10% glucose, respectively, compared to the basal level of H2O2 production (i.e., 0.33 mM) by WT EcN. However, the bacterial growth was restricted possibly because of the oxidative stress of H2O2, further confirming the function of SpxB for H2O2 production.
To determine the H2O2 production, we tested the transformed E. coli Nissle carrying ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (GM EcN) in the culture medium of LB supplemented with 5% or 10% glucose. The supernatants of overnight culture were subjected to H2O2 production measurement. The assay showed that 2.15 mM and 1.84 mM of H2O2 per OD600 were produced by GM EcN cultured in 5% and 10% glucose, respectively, compared to the basal level of H2O2 production (i.e., 0.33 mM) by WT EcN. However, the bacterial growth was restricted possibly because of the oxidative stress of H2O2, further confirming the function of SpxB for H2O2 production.
Fig. 5. The H2O2 was measured with a Fluorimetric Hydrogen Peroxide Assay Kit (SIGMA-ALDRICH). The H2O2 was produced by the transformants expressing AQP and SpxB.
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.
INDUCIBLE PROMOTER
The tpxp (thiol peroxidase promoter) is a H2O2-responsive promoter in S. mutans, which has been examined in the research by Jessica K. Kajfasz, et al. Thiol peroxidase catalyzes the reduction of hydrogen peroxide to water. It plays a role in cell protection against oxidative stress. The gene expression is highly correlated to the concentration of H2O2.
According to Jessica K. Kajfasz’s study of transcriptome responses of S. mutans to peroxide stress, the DNA fragments of 6 H2O2-responsive promoter elements with intrinsic RBS were amplified from gDNA of S. mutans by PCR and cloned onto pSB1C3. After assembled with GFP-Tr/pSB1C3 and tested the promoter activities, SMU_924 (tpxp) has the best H2O2-regulated properties (data not shown), which is suitable for our study. The basic part of tpxp/pSB1C3 was documented in Part Registry as BBa_K3376003. It’s further assembled with a reporter, GFP-Tr/pSB1C3 (BBa_K3376001), with or without the expression of AQP by ldhp, to generate tpxp-GFP-Tr/pSB1C3 (BBa_K3376004) and ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 (BBa_K3376007), respectively. All inserted sequences on the parts were confirmed by sequencing with VF2 and VR primes.
The tpxp (thiol peroxidase promoter) is a H2O2-responsive promoter in S. mutans, which has been examined in the research by Jessica K. Kajfasz, et al. Thiol peroxidase catalyzes the reduction of hydrogen peroxide to water. It plays a role in cell protection against oxidative stress. The gene expression is highly correlated to the concentration of H2O2.
According to Jessica K. Kajfasz’s study of transcriptome responses of S. mutans to peroxide stress, the DNA fragments of 6 H2O2-responsive promoter elements with intrinsic RBS were amplified from gDNA of S. mutans by PCR and cloned onto pSB1C3. After assembled with GFP-Tr/pSB1C3 and tested the promoter activities, SMU_924 (tpxp) has the best H2O2-regulated properties (data not shown), which is suitable for our study. The basic part of tpxp/pSB1C3 was documented in Part Registry as BBa_K3376003. It’s further assembled with a reporter, GFP-Tr/pSB1C3 (BBa_K3376001), with or without the expression of AQP by ldhp, to generate tpxp-GFP-Tr/pSB1C3 (BBa_K3376004) and ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 (BBa_K3376007), respectively. All inserted sequences on the parts were confirmed by sequencing with VF2 and VR primes.
Fig. 7. The cloning of H2O2-responsive promoters of S. mutans, tpxp-GFP-Tr/pSB1C3 (BBa_K3376004) and ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 (BBa_K3376007). (A) The H2O2-responsive promoters were amplified as inserts by PCR with BioBrick prefix and suffix elements (554 bp and 1867 bp, respectively) from gDNA of S. mutans. (B) tpxp-GFP-Tr/pSB1C3 was checked by EcoRI and PstI (2029 + 1091 bp). (C) ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 was checked by EcoRI and PstI (2029 + 1767 bp).
To detect the basal level of promoter activity, E. coli Nissle with ldhp-GFP-Tr/pSB1C3 or tpxp-GFP-Tr/pSB1C3 were cultured overnight and the GFP was measured at Ex/Em = 483/513. The data indicated ldhp has high level of gene expression, while the activity of tpxp is limited.
To detect the basal level of promoter activity, E. coli Nissle with ldhp-GFP-Tr/pSB1C3 or tpxp-GFP-Tr/pSB1C3 were cultured overnight and the GFP was measured at Ex/Em = 483/513. The data indicated ldhp has high level of gene expression, while the activity of tpxp is limited.
Fig. 8. GFP was measured at Ex/Em = 483/513 and expressed at different levels with the indicated promoters. Inset plot: GFP fluorescence was observed under a blue LED box.
To investigate the promoter activity in response to H2O2, E. coli Nissle carrying ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 was treated with 3mM of H2O2 or not (as control). GFP level was increased significantly in the presence of H2O2 compared to the control group. Notably, there’s no H2O2 response in the absence of AQP, further confirming the H2O2 transport ability of AQP.
To investigate the promoter activity in response to H2O2, E. coli Nissle carrying ldhp-AQP-Tr-tpxp-GFP-Tr/pSB1C3 was treated with 3mM of H2O2 or not (as control). GFP level was increased significantly in the presence of H2O2 compared to the control group. Notably, there’s no H2O2 response in the absence of AQP, further confirming the H2O2 transport ability of AQP.
Fig. 9. GFP expression level was increased with AQP in the presence of H2O2 compared to the control without AQP.
To further verify the H2O2-regulated response, the cells were cultured in the various concentrations of H2O2. Compared to basal level of tpxp activity, GFP intensity was enhanced with increasing concentrations of H2O2 in a dose-dependent manner. Taken together, H2O2 may enter across cell membrane through AQP and induce tpxp promoter activity.
To further verify the H2O2-regulated response, the cells were cultured in the various concentrations of H2O2. Compared to basal level of tpxp activity, GFP intensity was enhanced with increasing concentrations of H2O2 in a dose-dependent manner. Taken together, H2O2 may enter across cell membrane through AQP and induce tpxp promoter activity.
Fig. 10. The promoter activity of tpxp was increased dose-dependently with H2O2 concentration in the presence of AQP.
ANTITOXIN GENE
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. The existing part of KatG-Tr/pSB1C3 (BBa_S04059) was created by iGEM team Caltech in 2008, demonstrating the function of KatG (AHL-inducible KatG expression device, BBa_K137079) in minimum inhibitory concentration (MIC) assay to revive cells from H2O2 shock.
We collected the KatG as our antitoxin gene and amplified from KatG/pSB1C3 by PCR. The resulting PCR product was assembled with the H2O2-regulated promoter of tpxp (tpxp-KatG-Tr/pSB1C3, BBa_K3376011) and further inserted into ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa_K3376010) to generate our final device, the composite part of ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 (BBa_K3376012).
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. The existing part of KatG-Tr/pSB1C3 (BBa_S04059) was created by iGEM team Caltech in 2008, demonstrating the function of KatG (AHL-inducible KatG expression device, BBa_K137079) in minimum inhibitory concentration (MIC) assay to revive cells from H2O2 shock.
We collected the KatG as our antitoxin gene and amplified from KatG/pSB1C3 by PCR. The resulting PCR product was assembled with the H2O2-regulated promoter of tpxp (tpxp-KatG-Tr/pSB1C3, BBa_K3376011) and further inserted into ldhp-AQP-RBS-SpxB-Tr/pSB1C3 (BBa_K3376010) to generate our final device, the composite part of ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 (BBa_K3376012).
Fig. 11. The cloning of ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 (BBa_K3376012). (A) The KatG was amplified as an insert by PCR with BioBrick prefix and suffix elements (2222 bp) from a BioBrick part (BBa_S04059). (B) Colony PCR check for ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3 with SpxB-forward primer and tpxp-reverse primer (2114 bp). (C) Restriction enzyme check for clones no. 1 and 3 by EcoRI and PstI (5021 + 2044 bp).
To characterize the function of the existing part of KatG-Tr/pSB1C3 (BBa_S04059), we tested the recovery activity in cell growth inhibition by H2O2 production of AQP-SpxB in the presence or absence of KatG. As shown in our data, 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.
To characterize the function of the existing part of KatG-Tr/pSB1C3 (BBa_S04059), we tested the recovery activity in cell growth inhibition by H2O2 production of AQP-SpxB in the presence or absence of KatG. As shown in our data, 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. 12. Growth recovery and the function of KatG. 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.
Furthermore, although expression of KatG, the H2O2 production rate was not affected in the high concentration of 10% glucose. However, in the lower concentrations of glucose, the KatG may decompose the H2O2 production by SpxB. It’s reasonable because H2O2 production rate by SpxB is correlated to the level of glycolysis, which is response to the concentration of glucose. Furthermore, the phenomenon confirms the H2O2-regulated properties of tpxp activity.
Furthermore, although expression of KatG, the H2O2 production rate was not affected in the high concentration of 10% glucose. However, in the lower concentrations of glucose, the KatG may decompose the H2O2 production by SpxB. It’s reasonable because H2O2 production rate by SpxB is correlated to the level of glycolysis, which is response to the concentration of glucose. Furthermore, the phenomenon confirms the H2O2-regulated properties of tpxp activity.
Fig. 13. 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.
PROTEIN EXPRESSION OF THE PARTS
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.
