Part:BBa_K3002103
Level 1 – Wildtype PETase + His (Phytobrick)
This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), a HA-tag (BBa_K3002017) and the coding sequence of the wildtype PETase (BBa_K3002004) 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. To purify PETase and to differentiate between MHETase and PETase we designed a His-tag. Since Mut-PETase is essential for the degradation of PET and showed activity against PET and BHET we also designed PETase without any mutations to compare enzymatic activity. So we tagged Wildtype-PETase with His-tag.
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 1002
Illegal PstI site found at 1975 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1002
Illegal PstI site found at 1975 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1002
Illegal PstI site found at 1975 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1002
Illegal PstI site found at 1975
Illegal NgoMIV site found at 737
Illegal NgoMIV site found at 764
Illegal NgoMIV site found at 2415 - 1000COMPATIBLE WITH RFC[1000]
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