Part:BBa_K3002037
Wildtype MHETase for Chlamydomonas reinhardtii (Phytobrick)
This basic part contains the coding sequence of the wildtype MHETase (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii. It is a fusion of the parts BBa_K3002029 and BBa_K3002005. In combination with a promoter and a terminator, this level 0 construct mediates MHET degradation ability. As this part contains the introns 1 and two times intron 2 of RBCS2, it perfectly matches the part BBa_K3002027 (pAR promoter A1-B2), resulting in a high expression (Eichler-Stahlberg et al., 2009). To detect or purify the target protein a tag of the Kaiser Collection like BBa_K3002010 (sp20 HA-tag), BBa_K3002017 (HA-tag), BBa_K3002018 (sp20 His-tag), BBa_K3002028 (His-tag) is recommended. A secretion signal (BBa_K3002007 (cCA), BBa_K3002008 (GLE) or BBa_K3002009 (ARS)) can be added, when using the part BBa_K3002003 (pAR promoter (A1-A3)) as a promoter.
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Effect of the SP20 module on the secretion efficiency of MHETase. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with different secretion signals and a C-terminal SP20 tag for enhancing glycosylation. (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 (BBa_K3002202) and A27 (BBa_K3002200) served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.
Identification of MHETase and MUT-PETase by LC-MS/MS. (a) Transformants generated with construct L2N (d) (BBa_K3002213) 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. Protein bands corresponding to those detected with the anti-HA antibody in a gel run in parallel and stained with Coomassie brilliant blue were excised, in-gel digested with trypsin and analyzed by LC-MS/MS. Peptides identified by LC-MS/MS for MHETase (green) and MUT-PETase (purple) are indicated. (b, c) Sequences of MHETase and MUT-PETase with the peptides detected by LC-MS/MS are highlighted in green and purple, 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 765
Illegal PstI site found at 1089
Illegal PstI site found at 1432
Illegal PstI site found at 2242 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 765
Illegal PstI site found at 1089
Illegal PstI site found at 1432
Illegal PstI site found at 2242
Illegal NotI site found at 1100 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2010
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 765
Illegal PstI site found at 1089
Illegal PstI site found at 1432
Illegal PstI site found at 2242 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 765
Illegal PstI site found at 1089
Illegal PstI site found at 1432
Illegal PstI site found at 2242
Illegal NgoMIV site found at 698
Illegal NgoMIV site found at 1159
Illegal NgoMIV site found at 1189
Illegal NgoMIV site found at 1504
Illegal NgoMIV site found at 1522
Illegal NgoMIV site found at 1558
Illegal NgoMIV site found at 2201 - 1000COMPATIBLE WITH RFC[1000]
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