Part:BBa_K3002124
Level 1 � Wildtype-PETASE + cCA + sp20-His
This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), the SP20-His-tag (BBa_K3002018), the secretion signal cCA (BBa_K3002007) and the coding sequence of the Wildtype-PETase (BBa_K3002004) plus the MoClo connectors for positions B1-B2 (BBa_K3002302), B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).
Since the Mut-PETase in combination with the secretion signal cCA, the SP20-module and HA-tag showed secretion by C.reinhardtii we designed this construct harboring a His-tag instead of a HA-tag. His-tag is designed to allow purification of MUT-PETase via Ni-NTA column and to differentiate between HA-tagged MHETase and His-tagged MUT-PETase. Because MUT-PETase showed activity against PET and BHET we wanted to compare the enzymatic activity of MUT-PETase with PETase without any mutations (Wildtype-PETase) and therefore designed the construct with wild-type PETase.
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 1069
Illegal PstI site found at 2042 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1069
Illegal PstI site found at 2042 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1069
Illegal PstI site found at 2042 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1069
Illegal PstI site found at 2042
Illegal NgoMIV site found at 804
Illegal NgoMIV site found at 831
Illegal NgoMIV site found at 2533 - 1000COMPATIBLE WITH RFC[1000]
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