Part:BBa_K3002005
Wildtype MHETase for Chlamydomonas reinhardtii Part 1 (Phytobrick)
This basic part contains the coding sequence of the wildtype MHETase (B3). This part is codon-optimized for Chlamydomonas reinhardtii. Combined with the second part of the MHETase (BBa_K3002029), a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the introns 1 and 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.
This part was designed to allow synthesis due to a high GC-level. It is only functional in combination with the second part (BBa_K3002029), of the MHETase. Visit the page of the part BBa_K3002037 to gain a deeper understanding of the functionality of the MHETase
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 711
Illegal PstI site found at 1089 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 711
Illegal PstI site found at 1089
Illegal NotI site found at 1100 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 711
Illegal PstI site found at 1089 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 711
Illegal PstI site found at 1089
Illegal NgoMIV site found at 1027
Illegal NgoMIV site found at 1159
Illegal NgoMIV site found at 1189 - 1000COMPATIBLE WITH RFC[1000]
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