Part:BBa_K3002105
Level 1 – Mutant PETase + HA (Phytobrick)
This composite part contains the PAR-promoter of the team iGEM17_NU_Kazakhstan (BBa_K2516001) in combination with the RPL23-Terminator (BBa_K3002006), a HA-tag (BBa_K3002017) and the coding sequence of the mutant PETase (BBa_K3002014) plus the MoClo connectors for positions B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).
Both MUT-PETase and MHETase were strongly expressed in the cytosol by C. reinhardtii. The Mut-PETase is essential for the degradation of PET and showed activity against PET and BHET.
Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii. (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). (b) The UVM4 strain was transformed with the construct shown in (a). 11 spectinomycin-resistant transformants were inoculated in TAP and samples taken after 3 days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW – molecular weight. The black arrow represents the MHETase, the white arrow the MUT-PETase. The expression of both MHETase (~70 kDa) and MUT-PETase (~35 kDa) is visible in colonies 18, 22 and 27. The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.
The Chlamy Yummy Project Collection
We are proud to present our MoClo part collection for C. reinhardtii - the Chlamy Yummy project collection.
These 67 parts are all parts used during our project and were specifically designed and codon optimized for Chlamydomonas. Among them are basic parts (L0) of a novel mutant of the PETase (BBa_K3002014), the wildtype PETase and MHETase as well as a variety of functional composite parts (L1+2). Containing different tags as well as selection markers, this collection serves as a perfect base for plastic degradation projects with Chlamydomonas. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. Because this is a MoClo collection, the parts are highly standardized for worldwide application. The combination with other part collections works fast and easy. While in MoClo, nomenclature is a bit different from the iGEM BioBricks, it is quickly explained:
Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.
Level 1 parts are combinations of basic parts and usually form functional transcription units.
Level 2 parts are combinations of Level 1 parts, in case you want to transfer multiple transcription units at once. For example, you can pair your gene of interest with a selection marker.
The great thing about the Kaiser Collection and MoClo is that the ligation works in a one pot, one step reaction, as the Type IIs restriction enzymes cut out their own recognition sites. This way, multiple constructs can be combined linearly in a fixed order to create complex structures. This is ensured by the standardized overlaps that assign the parts one of 10 positions in the final constructs. After trying MoClo once, you won’t go back to traditional ligation. It is incredibly easy and reliable. Visit our parts site to get an overview over all parts.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1142
Illegal PstI site found at 2115 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 264
Illegal PstI site found at 1142
Illegal PstI site found at 2115 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1142
Illegal PstI site found at 2115 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1142
Illegal PstI site found at 2115
Illegal NgoMIV site found at 877
Illegal NgoMIV site found at 904
Illegal NgoMIV site found at 2555 - 1000COMPATIBLE WITH RFC[1000]
| None |