Team:Nanjing-China/Demonstrate
Overview
The following list exhibits our key achievements in this project. Our design successfully works out under real conditions.
- Achieved aerobic hydrogen production using our design.
- Constructed and Tested recombinant with hydrogenase.
- Constructed and Tested light-driven system based on OmpA-PbrR protein.
- Constructed desired structure of silicon encapsulation system.
- Tested silicon encapsulation system with stability and viability.
Our design consists of three modules: hydrogenase, artificial PS system, silicon encapsulation. All three modules have gone through examination:
Hydrogenase
Overview
Hydrogenase is defined as the enzyme that catalyzes the reversible oxidization of hydrogen. Our target enzyme EcHyd-1 is encoded by a six-gene operon hyaABCDEF on the genome of E.coli. To achieve hydrogen production in E.coli, we induced overexpression of this native hydrogenase. All six genes will also be our Parts this year.
Key achievements:
- Constructed recombinant plasmid pET-28a with hyaABCDEF under T7 promoter
- Succeeded in induced overexpression of hydrogenase in recombinant E.coli (Figure A, B)
- Conducted qualitative proof of the efficiency of hydrogenase using redox indicator WO3 (Figure C)
- Conducted quantitative proof of the efficiency of hydrogenase using gas chromatography
- Completed standard curve of bulk H2 to measure the amount of H2 in samples (Figure D)
- Achieved double H2 production over native fermentation: in 600mL head space after 20h incubation: 14.71μmol in recombinant group against 6.88μmol in control group (Figure E)
- Completed standard curve of bulk H2 to measure the amount of H2 in samples (Figure D)
- Achieved double H2 production over native fermentation: in 600mL head space after 20h incubation: 14.71μmol in recombinant group against 6.88μmol in control group (Figure E)
PbrR-based Artificial PS System
Overview
The key element of light driven system in our design is induce-precipitated CdS semiconductor nanoparticles, constructed by OmpA-PbrR fused protein. Semiconductors, similar to natural PS systems, provide excited electrons under illumination. These electrons are then passed down to redox dye methyl viologen (MV) and eventually hydrogenases.
Key achievements:
- Induced precipitation of in situ CdS particles
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Also have a modeling dealing with Cd2+ sewage problems
- Confirmed photocatalytic reduction capability of methyl viologen using induce-precipitated of CdS particles on E.coli cells. (Figure A)
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Positive reference: TiO2 semiconductor nanoparticles
- Confirmed compatibility of CdS particles and hydrogenase. (Figure B)
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Achieved improved H2 production: in 123mL head space after 20h incubation: 1.22 with CdS particles against 0.98μmol in control group
- Obtained TEM images of bacteria and confirmed particles smaller than 10nm. (Figure C)
- Also have a modeling dealing with Cd2+ sewage problems
- Positive reference: TiO2 semiconductor nanoparticles
- Achieved improved H2 production: in 123mL head space after 20h incubation: 1.22 with CdS particles against 0.98μmol in control group
Silicon Encapsulation
Overview
Hydrogenase is very sensitive to oxygen. To overcome the oxygen sensitivity, we designed and tested our layer-by-layer encapsulation method and achieved desired micro-ball structure. We assume spatial functional differentiation occurred within the system where the exterior consumes oxygen and creates an anaerobic core. Not only is the closed system long-term stable, but also it keeps living bacteria with viability.
Key achievements:
- Confirmed at least 12h stability of encapsulation with E.coli growth curves (Figure A)
Also have a modeling part to simulate and predict growth curves (Figure B1, B2)
- Confirmed viability of living bacteria within closed system according to a water-culture 20-day fluorescence decay experiment (Figure C)
- Confirmed desired micro-ball structure using fluorescence microscope (Figure D1, D2)
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Also have a modeling part to simulate and predict growth curves (Figure B1, B2)
Whole Cell H2 Production
Overview
In real conditions we combined all the three modules to build a whole cell H2 in air factory. Our design successfully works out under this condition.
Key achievements:
- Confirmed successful hydrogen production using our hydrogenase, CdS system and encapsulation shell under aerobic culture. (Figure A)
- Also have a modeling part for hydrogen diffusion and theoretical hydrogen-oxygen balance. (Figure B1, B2)
- 16 groups of experiment with four variables (particles, reaction solution, oxygen level, encapsulation or not) as back up to ensure effectiveness of every module
With three modules: hydrogenase, light driven system, encapsulation system and combined design all tested, the above confirms success try-out of our design under real conditions.
Further experiments will be conducted on:
- Replication of photocatalytic reduction assay of CdS particles
- Replication and improvement on whole cell H2 factories
- TEM images of silicon encapsulated microballs to discover exact inner structures
- Fitting of hydrogen-oxygen concentration theoretical model with experiment data.
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CONTACT
Address
- Life Science Department
- #163 Xianlin Blvd, Qixia District
- Nanjing University
- Nanjing, Jiangsu Province
- P.R. of China
- Zip: 210046
- nanjing_china@163.com
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SPONSORS
南京大学
生命科学学院
SCHOOL OF LIFE SCIENCES
NANJING UNIVERSITY
南京大学
NANJING UNIVERSITY
南京大学
化学化工学院
SCHOOL OF CHEMISTRY AND CHEMICAL ENGINEERING
NANJING UNIVERSITY
