Part:BBa_K3002107
Level 1 – Mutant PETase + GLE + HA (Phytobrick)
This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), GLE secretion signal (BBa_K3002008), HA-tag (BBa_K3002017) and the coding sequence of the mutant PETase (BBa_K3002014) plus the MoClo connectors for positions B1-B2 (BBa_K3002302), B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).
Construct is secreted trough secretion signal GLE. The secretion signal GLE shows minor secretion of the protein without combination with SP20 tag. Construct containing cCA leads to very low yield of the secreted protein in comparison to the other secretion signals.
Analysis of the secretion of MUT-PETase with secretion signals cCA, GLE, and ARS. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase equipped with secretion signals from carbonic anhydrase (cCA), gamete lytic enzyme (GLE) and arylsulfatase (ARS). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs shown in (a) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody.Transformants C12 and A27 introduced in Figures 4 and 5, respectively, served as positive controls. The white arrow points to the MUT-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 1087
Illegal PstI site found at 2060 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1087
Illegal PstI site found at 2060 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1087
Illegal PstI site found at 2060 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1087
Illegal PstI site found at 2060
Illegal NgoMIV site found at 822
Illegal NgoMIV site found at 849
Illegal NgoMIV site found at 2500 - 1000COMPATIBLE WITH RFC[1000]
| None |