Team:Aalto-Helsinki/LabResults
Laboratory Results
The constructs
Overview
Our goal was to produce propane from cellulose in E.coli. To achieve this our aim was to build three plasmids and transform them to the same bacteria. Two of the plasmids include the ten genes necessary for propane production in E.coli. The third plasmid contains the three genes encoding the enzymes for cellulose hydrolysis. To ease the bottlenecks caused by two of the enzymes in the propane pathway we built an amphiphilic protein biobrick. To validate our amphiphilic protein we created a new, fusable GFP biobrick. Amphiphilic micelle formation was validated with electron microscopy.
Propane producing plasmid 1: CAR
CAR is the first of our propane producing plasmids. The plasmid was constructed to pSB6C1 backbone with Gibson assembly. Afterwards the CAR insert was transferred to the biobrick backbone pSB1C3 with restriction digestion and ligation.
Achievements:
- Gibson assembly with pSB6A1 backbone successful according to colony-PCR
- Successfully transferred CAR construct to biobrick backbone pSB1C3
Propane producing plasmid 2: AtoB
AtoB is the second of our propane producing plasmids. The plasmid was straight built to the biobrick backbone pSB1C3 with Gibson assembly.
Achievements:
- Gibson assembly with biobrick backbone pSB1C3 successful according to restriction analysis
Amphiphilic protein
Ampihilic protein was created to fuse the two bottleneck enzymes of our propane pathway close to each other.
Achievements:
- Amphiphilic protein biobrick was sequenced successfully
Fusable GFP
To help testing our amphiphilic protein we built a fusable GFP biobrick. The fusable GFP biobrick has one additional nucleotiode prior to it’s suffix: it can be fused with any aminoacid’s amino terminus without losing it’s reading frame.
Achievements:
- Created a fusable GFP biobrick
- Fusable GFP was sequenced successfully
- Successfully fused the fusable GFP biobrick with ampiphilic protein