Team:Pasteur Paris/Parts
Parts
BBa_K1622000
Part Composition
This part is composed of 3 subparts :
- RBS: Ribosome Binding Site
- Glycoaldehyde Dehydrogenase
- Glycoaldehyde Reductase
Glycoaldehyde Dehydrogenase
This enzyme catalyses the Break-down of Glycoaldehyde into Glycolate, which is the third reaction of our PET degradation chain. We obtained the original sequence of this subpart in the iGEM registry of standard parts (BBa_936011). We optimized our sequence for E.coli and ordered the synthesis from our sequence.
Glycoaldehyde Reductase
The ethylene glycol to glycoaldehyde reaction is catalyzed by this enzyme. In the same manner as the Glycoaldehyde Dehydrogenase, we got the original sequence on the registry (BBa_K936023), optimized those sequences for E.coli and ordered their synthesis.
Part Assembly
The subparts were assembled using Gibson Assembly. Prior to the actual assembly, we had to amplify our sequences via PCR using primers with extensions that contain overlapping sequences.
BBa_K1622001
Part Composition
This part is a composite part composed of 2 Biobricks :
- BBa_K808030: NB-Esterase
- BBa_K808007: TPA Transporter
NB-Esterase
This enzyme catalyses the degradation of PET into Terephthalique acid and Ethylene Glycol. It is the first and slowest reaction of our degradation chain. Indeed, it takes two weeks to degrade PET.
We performed an enzymatic assay of NB-Esterase using 4-nitrophenylbutyrate as a substrate because it has a close chemical structure to PET and we figured that this enzyme digested pNP in our bacteria
TPA Transporter
The NB-Esterase is present in the extracellular medium and cuts PET in this location. In order to be fully degraded, the TPA needs to be integrated to our chassis organism which is why we incorporated a transporter to our degradation operon.
Part Assembly
The subparts were assembled using standard BioBrick Assembly.
^
Page up