Part:BBa_K3002227
L2 hygromycin resistance + ccA_Mut-PETase_SP20HA
This composite part contains a hygromycin resistance (BBa_K3002119) and the mutant PETase with the cCA (BBa_K3002109) fused with an SP20HA-tag for easy detection via HA-antibody and enhanced secretion.
The construct encodes the secretion signal cCA in front of the MUT-PETase gene and an SP20-tag behind it. As selection marker a hygromycin cassette is used. Since it is the additional construct in a co-transformation, it must have a different selection marker than the already existing construct, which is an aadA cassette. The MUT-PETase and MHETase are both seen after the co-transformation. An ARS secretion signal is upstream to the MUT-PETase gene and the cCA secretion signal upstream to the MHETase gene. The MUT-PETase is crucial for the degradation of PET into terephthalic acid and ethylene glycol.
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 EcoRI site found at 1789
Illegal PstI site found at 2873
Illegal PstI site found at 3846 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1789
Illegal NheI site found at 2053
Illegal PstI site found at 2873
Illegal PstI site found at 3846 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1789
Illegal BamHI site found at 1408
Illegal XhoI site found at 250 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1789
Illegal PstI site found at 2873
Illegal PstI site found at 3846 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1789
Illegal PstI site found at 2873
Illegal PstI site found at 3846
Illegal NgoMIV site found at 773
Illegal NgoMIV site found at 955
Illegal NgoMIV site found at 1326
Illegal NgoMIV site found at 2608
Illegal NgoMIV site found at 2635
Illegal NgoMIV site found at 4406 - 1000COMPATIBLE WITH RFC[1000]
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