Molecular Experiment
6.30-7.1
Transform plasmids PUC57-slr0168(vector) and PUC57-slr2030(vector) to E. coli TOP10.
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
Inoculate E. coli PUC57-slr0168(vector), PUC57-slr2030(vector).
Fig 1. Left: The PCR identification result of PUC57-slr0168(vector)
Right: The PCR identificationotebn result of PUC57-slr2030(vector)
Purification of fragments PUC57-slr0168(vector) and PUC57-slr2030(vector)
Preparation of E. coli WM3064 competent cell.
7.3-7.4
Transform plasmids ldhA-lldP-omcs to E. coli TOP10.
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
Inoculate E. coli ldhA-lldP-omcs.
Fig 2. Left: The PCR identification result of ldhA-lldP-omcs
Right: The PCR identification result of ldhA-lldP-omcs
Purification of the fragments ldhA-lldP-omcs and omcs.notebook
7.6-7.7
Transform plasmids (PYYDT(vector), ycel-pncB and nadE-nadD-nadM) to E. coli TOP10.
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
Inoculate E. coli PYYDT(vector), ycel-pncB and nadE-nadD-nadM.
7.8
Extract plasmids (PYYDT(vector), ycel-pncB and nadE-nadD-nadM).
PCR and electrophorese to confirm.
Fig 3. Left: The PCR identification result of PYYDT (vector)
Right: The PCR identification result of ycel-pncB and nadE-nadD-nadM
Purification of the fragments PYYDT(vector), ycel-pncB and nadE-nadD-nadM.
7.9
SOE PCR of ycel-pncB and nadE-nadD-nadM, and electrophorese to confirm.
Fig 4. The PCR identification result of ycel-pncB and nadE-nadD-nadM
Purification of the fragments ycel-pncB and nadE-nadD-nadM.
7.10-7.11
Transform plasmids (dsup-gshA-gshB) to E. coli TOP10.
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
Inoculate E. coli dsup-gshA-gshB.
7.12
Extract plasmids (dsup-gshA-gshB).
PCR and electrophorese to confirm.
Fig 5. The PCR identification result of dsup-gshA-gshB
Purification of dsup-gshA-gshB.
7.13-7.14
Transform plasmids (dsup-sodA-oprf) to E. coli TOP10.
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
Inoculate E. coli dsup-sodA-oprf.
7.15
Extract plasmids (dsup-sodA-oprf).
PCR and electrophorese to confirm.
Fig 6. The PCR identification result of dsup-sodA-oprf
Purification of dsup-sodA-oprf.
We have obtained all the plasmids and fragments we need and will next attempt to construct plasmids using homologous recombination.
7.16
Homologous recombination of ldhA-lldP-omcs and PUC57-slr0168(vector).
Homologous recombination of omcs and PUC57-slr0168(vector).
Transform the plasmids (homologous recombination) to E. coli TOP10.
7.17
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
We didn’t succeed in connecting the fragments.
7.18
Homologous recombination of ldhA-lldP-omcs and PUC57-slr2030(vector).
Transform the plasmids (homologous recombination) to E. coli TOP10.
7.19
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
We didn’t succeed in connecting the two fragments. It was suspected that there was a problem with the design of the primer homology arm and the primers were redesigned.
7.20-7.21
Inoculate E. coli ldhA-lldP-omcs and PUC57-slr0168(vector).
Extract plasmids ldhA-lldP-omcs and PUC57-slr0168(vector).
PCR with redesigned primers and electrophorese to confirm.
Fig 7. Left: The PCR identification result of ldhA-lldP-omcs
Middle: The PCR identification result of omcs
Right: The PCR identification result of PUC57-slr0168(vector)
Purification of fragments ldhA-lldP-omcs, omcs and PUC57-slr0168(vector).
7.22-7.23
Inoculate E. coli dsup-gshA-gshB and PUC57-slr2030(vector).
Extract plasmids dsup-gshA-gshB and PUC57-slr2030(vector).
PCR with redesigned primers and electrophorese to confirm.
Fig 8. Left: The PCR identification result of dsup-gshA-gshB
Right: The PCR identification result of PUC57-slr2030(vector)
Purification of fragments dsup-gshA-gshB and PUC57-slr2030(vector).
7.24-7.25
Inoculate E. coli dsup-sodA-oprf, ycel-pncB, nadE-nadD-nadM and PYYDT(vector)
Extract plasmids dsup-sodA-oprf, ycel-pncB, nadE-nadD-nadM and PYYDT(vector).
PCR with redesigned primers and electrophorese to confirm.
Fig 9. Left: The PCR identification result of nadE-nadD-nadM and ycel-pncBycel-pncB
Middle: The PCR identification result of PYYDT(vector)
Right: The PCR identification result of dsup-sodA-oprf
Purification of fragments nadE-nadD-nadM, ycel-pncB, PYYDT(vector),and dsup-sodA-oprf.
7.26
SOE PCR of ycel-pncB and nadE-nadD-nadM, and electrophorese to confirm.
Fig 10. The PCR identification result of ycel-pncB and nadE-nadD-nadM
Purification of fragments ycel-pncB and nadE-nadD-nadM.
We have obtained all the plasmids and fragments we need by PCR with redesigned primers and will next attempt to construct plasmids using homologous recombination.
7.27
Homologous recombination of ldhA-lldP-omcs and PUC57-slr0168(vector).
Homologous recombination of omcs and PUC57-slr0168(vector).
Transform the plasmids (homologous recombination) to E. coli TOP10.
7.28
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
Fig 11. The PCR identification result of plasmid omcs (Ppsba2-omcs-Tpsbc)
Plasmid omcs (Ppsba2-omcs-Tpsbc) was constructed successfully. Combined with water and glycerol, preserve.
We didn't succeed in connecting the ldhA-lldP-omcs and PUC57-slr0168(vector).
7.29
Homologous recombination of dsup-gshA-gshB and PUC57-slr2030(vector).
Transform the plasmids (homologous recombination) to E. coli TOP10.
7.30
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
We didn’t succeed in connecting the dsup-gshA-gshB and PUC57-slr2030(vector).
7.31
Homologous recombination of ycel-pncB-nadE-nadD-nadM and PYYDT(vector).
Homologous recombination of ycel-pncB and PYYDT(vector).
Homologous recombination of nadE-nadD-nadM and PYYDT(vector).
Transform the plasmids (homologous recombination) to E. coli TOP10.
8.1
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
Fig 12. Left: The PCR identification result of plasmid ycel-pncB（Ptac-ycel-pncB-TrrnB T1）and nadE-nadD-nadM （Ptac-nadE-nadD-nadM-TrrnB T1）
Plasmid ycel-pncB（Ptac-ycel-pncB-TrrnB T1）and nadE-nadD-nadM （Ptac-nadE-nadD-nadM-TrrnB T1）were constructed successfully. Combined with water and glycerol, preserve.
8.2
Homologous recombination of dsup-sodA-oprf and PYYDT(vector.
Transform the plasmids (homologous recombination) to E. coli TOP10.
8.3
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
We didn’t succeed in connecting the dsup-sodA-oprf and PYYDT(vector).
Through homologous recombination, we constructed the plasmids omcs, ycel-pncB, nadE-nadD-nadM. For the remaining plasmids that were not constructed, we will try to construct them by double enzymes digestion and enzyme ligation.
8.4
Double enzymes digestion of ldhA-lldP-omcs and PUC57-slr0168(vector).
Ligating ldhA-lldP-omcs with PUC57-slr0168(vector).
Transform the plasmids (ligation) to E. coli TOP10.
8.5
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
Fig 13. The PCR identification result of plasmid pLactate (Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc）
Plasmid pLactate (Pcpc560-ldhA-lldP-Tpsbc-Ppsba2-omcs-Tpsbc）was constructed successfully. Combined with water and glycerol, preserve.
8.6
Double enzymes digestion of dsup-gshA-gshB and PUC57-slr2030(vector).
Ligating dsup-gshA-gshB with PUC57-slr2030(vector).
Transform the plasmids (ligation) to E. coli TOP10.
8.7
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
Fig 14. The PCR identification result of plasmid pResistance6803 (Pcpc560-dsup-gshA-gshB-Tpsbc-Ppsba2-slr2030-Tpsbc）
Plasmid pResistance6803 (Pcpc560-dsup-gshA-gshB-Tpsbc-Ppsba2-slr2030-Tpsbc）was constructed successfully. Combined with water and glycerol, preserve.
8.8
Ligating ycel-pncB-nadE-nadD-nadM with PYYDT(vector).
Transform the plasmids (ligation) to E. coli TOP10.
8.9
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
Fig 15. The PCR identification result of plasmid pElectricty（Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1）
Plasmid pElectricty（Ptac-ycel-pncB-TrrnB T1-Ptac-nadE-nadD-nadM-TrrnB T1）was constructed successfully. Combined with water and glycerol, preserve.
8.10
Double enzymes digestion of dsup-sodA-oprf.
Ligating dsup-sodA-oprf with PYYDT(vector).
Transform the plasmids (ligation) to E. coli TOP10.
8.11
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm.
Fig 16. The PCR identification result of plasmid pResistance MR-1（Ptac-dsup-sodA-oprf-TrrnB T1）
Plasmid pResistance MR-1（Ptac-dsup-sodA-oprf-TrrnB T1）was constructed successfully. Combined with water and glycerol, preserve.
The plasmids pLactate, omcs, pElectricty, nadE-nadD-nadM, ycel-pncB and pResistance MR-1 have been constructed and transformed into E.coil TOP10. Next the plasmids pElectricty, nadE-nadD-nadM, ycel-pncB with pResistance MR-1 will be transformed into E.coil WM3064.
8.12-8.13
Inoculate E. coli pElectricty (TOP10), nadE-nadD-nadM (TOP10), ycel-pncB (TOP10).
Extract plasmids（pElectricty、nadE-nadD-nadM、ycel-pncB）.
Transform plasmids (pElectricty、nadE-nadD-nadM、ycel-pncB) to E. coli WM3064.
8.14
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
8.15-8.16
Inoculate E.coli pResistance MR-1 (TOP10).
Extract plasmids (pResistance MR-1).
Transform plasmids (pResistance MR-1) to E. coli WM3064.
8.17
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
8.18-8.19
Inoculate E.coli pResistance MR-1 (TOP10).
Extract plasmids (pResistance MR-1).
Transform plasmids (pResistance MR-1) to E. coli WM3064.
8.20
Pick the bacterial plaque. PCR bacterial fluid, and electrophorese to confirm. Combined with water and glycerol, preserve.
Expression Experiment
8.20-8.23
Transfer the plasmids pElectricty、ycel-pncB、nadE-nadD-nadM into Shewanella oneidensis MR-1 cells separately by conjugating with E.coli WM3064.
8.24-8.25
Pick the bacterial plaque and expand the Shewanella oneidensis MR-1 after conjugal transfer.
8.26
Cell disruption of wild-type and target gene (pElectricty、ycel-pncB、nadE-nadD-nadM) introduced Shewanella oneidensis MR-1.
8.27
Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the broken crude protein solution, and the possibility of electricity generation was preliminarily verified. It was found that the protein was successfully expressed except nadE-nadD-nadM.
8.27-8.28
Transfer the plasmids (nadE-nadD-nadM) into Shewanella oneidensis MR-1 cells by conjugating with E.coil WM3064.
8.29
Pick the bacterial plaque and expand the Shewanella oneidensis MR-1 after conjugal transfer.
8.30
Cell disruption of wild-type and target gene (nadE-nadD-nadM) introduced Shewanella oneidensis MR-1.
Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the broken crude protein solution, and the possibility of electricity generation was preliminarily verified. It was found that nadE nadD nadM was successfully transformed.
8.30-9.10
Transfer genes pLactate, omcs, and pResistance6803 into Synechocystis sp. PCC 6803and perform transformation validation. Expand the successfully transformed Synechocystis sp. PCC 6803.
9.10
Cell disruption of wild-type and target gene introduced Synechocystis sp. PCC 6803.
9.11
Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the broken crude protein solution.
9.12-9.13
Measure the lactate production of Synechocystis sp. PCC 6803 with the target gene, then organize and analyze the data.
9.13-9.15
Determination of NADH and NADH/NAD+in Synechocystis sp. PCC 6803 with different plasmids and target gene introduced Shewanella oneidensis MR-1.
9.16-9.18
Measure the electricity production ability of Shewanella oneidensis MR-1 at different rotational speeds, temperatures, and bacterial concentrations after conjugation transfer.
9.19-9.21
Add fluorescent dyes such as riboflavin to Shewanella oneidensis MR-1, and retest NADH and NADH/NAD+, as well as electricity production capacity.
9.21-9.28
Measure the OD600 of Shewanella oneidensis MR-1 and OD730 of Synechocystis sp. PCC 6803 introduced with different target genes, and plot their growth curves.
9.21-9.28
Configure Mbg11 culture medium to cultivate Shewanella oneidensis MR-1 and determine the growth curve of Shewanella oneidensis MR-1.
9.28-10.4
Explore the co cultivation conditions of Synechocystis sp. PCC 6803 and Shewanella oneidensis MR-1 (first analyze whether there is growth inhibition or toxicity to each other, and then explore the electricity production ability of the two under different proportions of inoculation), and draw their growth curves.
