Team:HUST-China/Experiments
Experiments
Part1: Photosynthetic microorganism system
1.1 Synechocystis sp.
1.1.1 Plasmid Construction in E.coli
We constructed following vectors carrying our parts and transformed these vectors into corresponding host strains.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-ldhD-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-ldhDC-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-ldhDnARSdR-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-ldhDARSdR-RBS-lldP | pSB1C3 | E.coli DH5α |
| RBS-TH-RBS-gldA-RBS-lldp | pSB1C3 | E.coli DH5α |
1.1.2 Add Flag Sequence into parts
The constructed plasmid was transformed into E.coli Top 10.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-ldhD-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDC-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDnARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |
1.1.3 Add 6×His Sequence into parts
The constructed plasmid was transformed into E.coli Top 10.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-ldhD-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDC-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDnARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |
| RBS-ldhDARSdR-RBS-lldP | pSB1C3 | E.coli Top 10 |
1.1.4 pCK360 Plasmid Conjugation
We constructed following vectors carrying our parts and transformed these vectors into E.coli Top 10 to transformed into cynobacteria.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-ldhD-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
| RBS-ldhDC-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
| RBS-ldhDnARSdR-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
| RBS-ldhDARSdR-RBS-lldP | pck306 | E.coli Top 10; cynobacteria |
Figure 6. modified circuit of ldhD-lldP.
Figure 7. modified circuit of ldhDC-lldP.
Figure 8. modified circuit of ldhDnARSdR-lldP.
Figure 9. modified circuit of ldhDARSdR-lldP.
1.1.5 Detection of yellow fluorescent protein
Since plasmid pCK306 has yellow fluorescent protein sequence, we cultivated the engineering cynobacteria induced by rhamnose for 24h. And put them under the fluorescence microscope at the wave of 488nm excitation light
1.2 Rhodopseudomonas palustris
1.2.1 Plasmid Construction in E.coli
We constructed following vectors carrying our parts and transformed these vectors into corresponding host strains.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-mleS | pSB1C3 | E.coli TOP10 |
| RBS-ldhA | pSB1C3 | E.coli TOP10 |
| RBS-lldP | pSB1C3 | E.coli TOP10 |
| RBS-mleS-RBS-lldP | pSB1C3 | E.coli TOP10 |
| RBS-lldP-RBS-ldhA-TT | pSB1C3 | E.coli TOP10 |
| RBS-ldhA-RBS-lldP-TT | pSB1C3 | E.coli TOP10 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pSB1C3 | E.coli TOP10 |
| RBS-mleS | pMG105 | E.coli TOP10 |
| RBS-ldhA | pMG105 | E.coli TOP10 |
| RBS-lldP | pMG105 | E.coli TOP10 |
| RBS-mleS-RBS-lldP | pMG105 | E.coli TOP10 |
| RBS-lldP-RBS-ldhA-TT | pMG105 | E.coli TOP10 |
| RBS-ldhA-RBS-lldP-TT | pMG105 | E.coli TOP10 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pMG105 | E.coli TOP10 |
1.2.2 Transformation into E.coli BL21
The constructed plasmid was transformed into E.coli BL21.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-mleS | pMG105 | E.coli BL21 |
| RBS-ldhA | pMG105 | E.coli BL21 |
| RBS-lldP | pMG105 | E.coli BL21 |
| RBS-mleS-RBS-lldP | pMG105 | E.coli BL21 |
| RBS-lldP-RBS-ldhA-TT | pMG105 | E.coli BL21 |
| RBS-ldhA-RBS-lldP-TT | pMG105 | E.coli BL21 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pMG105 | E.coli BL21 |
1.2.3 Transformation into Rhodopseudomonas palustris DSM5859
The constructed plasmid was transformed into Rhodopseudomonas palustris DSM5859.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-mleS | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-ldhA | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-lldP | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-mleS-RBS-lldP | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-lldP-RBS-ldhA-TT | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-ldhA-RBS-lldP-TT | pMG105 | Rhodopseudomonas palustris DSM5859 |
| RBS-mleS-RBS-ldhP-RBS-ldhA-TT | pMG105 | Rhodopseudomonas palustris DSM5859 |
1.2.4 Verification the expression at mRNA level
We did RT-qPCR to test the relative expression levels of each genes.
1.2.5 Detection of Lactate
We detect lactate by Lactic Acid assay kit.
Part2: Electrogenic microorganism system:
Shewanella oneidensis MR-1
2.1 Plasmid Construction in E.coli
We constructed following vectors carrying our parts and transformed these vectors into corresponding host strains.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-dld | pSB1C3 | E.coli TOP10 |
| RBS-lldEFG | pSB1C3 | E.coli TOP10 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pSB1C3 | E.coli TOP10 |
| RBS-gapA | pSB1C3 | E.coli TOP10 |
| RBS-mdh | pSB1C3 | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT | pSB1C3 | E.coli TOP10 |
| RBS-Ptac -RBS-pflB | pSB1C3 | E.coli TOP10 |
| RBS-fdh | pSB1C3 | E.coli TOP10 |
| RBS-pflB-RBS-fdh | pSB1C3 | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pSB1C3 | E.coli TOP10 |
| RBS-dld | pYYDT | E.coli TOP10 |
| RBS-lldEFG | pYYDT | E.coli TOP10 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pYYDT | E.coli TOP10 |
| RBS-gapA | pYYDT | E.coli TOP10 |
| RBS-mdh | pYYDT | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT | pYYDT | E.coli TOP10 |
| RBS-Ptac -RBS-pflB | pYYDT | E.coli TOP10 |
| RBS-pflB-RBS-fdh | pYYDT | E.coli TOP10 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pYYDT | E.coli TOP10 |
2.2 Transformation into E.coli WM3064
The constructed plasmid was transformed into a 2,6-diaminopimelic acid deficiency type E.coli WM3064.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-dld | pYYDT | pYYDT E .coli WM3064 |
| RBS-lldEFG | pYYDT | pYYDT E .coli WM3064 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pYYDT | pYYDT E .coli WM3064 |
| RBS-gapA | pYYDT | pYYDT E .coli WM3064 |
| RBS-mdh | pYYDT | pYYDT E .coli WM3064 |
| RBS-gapA-RBS-mdh-TT | pYYDT | pYYDT E .coli WM3064 |
| RBS-Ptac -RBS-pflB | pYYDT | pYYDT E .coli WM3064 |
| RBS-pflB-RBS-fdh | pYYDT | pYYDT E .coli WM3064 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pYYDT | pYYDT E .coli WM3064 |
2.3 Conjugation between E.coli and Shewanella oneidensis MR-1
We did conjugation after the transformation to WM3064 to introduce the plasmid into Shewanella oneidensis MR-1.
| Part | Plasmid Construction | Transformation to Host Strain |
|---|---|---|
| RBS-dld | pYYDT | Shewanella oneidensis MR-1 |
| RBS-lldEFG | pYYDT | Shewanella oneidensis MR-1 |
| RBS-dld-TT -Ptac -RBS-lldEFG | pYYDT | Shewanella oneidensis MR-1 |
| RBS-gapA | pYYDT | Shewanella oneidensis MR-1 |
| RBS-mdh | pYYDT | Shewanella oneidensis MR-1 |
| RBS-gapA-RBS-mdh-TT | pYYDT | Shewanella oneidensis MR-1 |
| RBS-Ptac -RBS-pflB | pYYDT | Shewanella oneidensis MR-1 |
| RBS-pflB-RBS-fdh | pYYDT | Shewanella oneidensis MR-1 |
| RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | pYYDT | Shewanella oneidensis MR-1 |
2.4 Verification the expression on mRNA level
We did RT-qPCR to test the relative expression levels of each genes.
2.5 Electrogenesis detection
We cultivated the engineering bacteria and put them into the electrogenesis device to test the output votage. We also did the measurement of electrode-attached biomass.
Co-culture and electricity
| Microorganism 1 | Microorganism 2 | Addition 1 | Addition 2 |
|---|---|---|---|
| E.coli | LB medium | ||
| Shewanella oneidensis MR-1 | LB medium | ||
| Shewanella oneidensis MR-1 | LB medium | Lactate | |
| Shewanella oneidensis MR-1 | Cynobacteria | LB medium | |
| Shewanella oneidensis MR-1 | Rhodopseudomonas palustris DSM5859 | LB medium | |
| Shewanella oneidensis MR-1（pYYDT- RBS-dld） | LB medium | Lactate | |
| Shewanella oneidensis MR-1(pYYDT-RBS-lldEFG） | LB medium | Lactate | |
| Shewanella oneidensis MR-1（pYYDT -RBS-dld-TT -Ptac -RBS-lldEFG） | LB medium | Lactate | |
| Shewanella oneidensis MR-1（pYYDT-RBS-gapA-RBS-mdh-TT ） | LB medium | Lactate | |
| Shewanella oneidensis MR-1（pYYDT -RBS-pflB-RBS-fdh） | LB medium | Lactate | |
| Shewanella oneidensis MR-1（pYYDT-RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB | LB medium | Lactate | Shewanella oneidensis MR-1（pYYDT- RBS-dld） | Rhodopseudomonas palustris DSM5859 | LB medium | Shewanella oneidensis MR-1（pYYDT-RBS-lldEFG） | Rhodopseudomonas palustris DSM5859 | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-dld-TT -Ptac -RBS-lldEFG） | Rhodopseudomonas palustris DSM5859 | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT ） | Rhodopseudomonas palustris DSM5859 | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-pflB-RBS-fdh） | Rhodopseudomonas palustris DSM5859 | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB） | Rhodopseudomonas palustris DSM5859 | LB medium | Shewanella oneidensis MR-1（pYYDT- RBS-dld） | Cynobacteria | LB medium | Shewanella oneidensis MR-1（pYYDT-RBS-lldEFG） | Cynobacteria | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-dld-TT -Ptac -RBS-lldEFG） | Cynobacteria | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT ） | Cynobacteria | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-pflB-RBS-fdh） | Cynobacteria | LB medium | Shewanella oneidensis MR-1（pYYDT -RBS-gapA-RBS-mdh-TT -RBS-Ptac -RBS-pflB） | Cynobacteria | LB medium |
Part3: Whole design
In this part, we mainly verify our vision by constructing MFC system and we detect the generated voltage. We did several parallel experiments to prove it:
1. Pre-experiment of electrogenesis.
MFC anode: Shewanella
MFC anode: Blank medium
MFC anode: E.coli
2. Lactate contrast.
MFC anode: Shewanella
MFC anode: Shewanella+lactate
3. Demonstrate that carbon cloth has higher electricity production efficiency than carbon rod.
MFC anode: carbon cloth
MFC anode: carbon rod
4. Study the effects of oxygen on Co-culture power systems.
MFC anode: anaerobic
MFC anode: aerobic
5. Contrast the symbiotic effect of wild-type strains
MFC anode: Synechocystis PCC6803 (wild type) + Shewanella
MFC anode: Rhodopseudomonas palustris (wild type) + Shewanella
6. Functional verification of engineered Rhodopseudomonas palustris
MFC anode: Rhodopseudomonas palustris (wild type) + Shewanella
MFC anode: Rhodopseudomonas palustris(engineered type) + Shewanella
7. Functional verification of engineered Synechococcus PCC6803
MFC anode: Synechocystis PCC6803 (wild type) + Shewanella
MFC anode: Synechocystis PCC6803 (engineered type) + Shewanella
