Part:BBa_K3002234
L2 hygromycin resistance 2 + cCA_Mut-PETase_SP20HA
This composite part contains a hygromycin resistance (BBa_K3002127) and the WT-PETase (BBa_K3002109) fused with an SP20HA-tag for easy detectionvia 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.
Analysis of secreted enzymes of transformant N6 transformed with construct AI. (b) Clones generated with transformant N6 (BBa_K3002213) and construct L2AI (a) (BBa_K3002234) were grown in TAP medium for four days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformant C12 (BBa_K3002202) served as positive controls. The black arrow points to MHETase, the white arrow to 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 3453
Illegal PstI site found at 4426 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2633
Illegal PstI site found at 3453
Illegal PstI site found at 4426 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3453
Illegal PstI site found at 4426 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3453
Illegal PstI site found at 4426
Illegal NgoMIV site found at 1270
Illegal NgoMIV site found at 1452
Illegal NgoMIV site found at 3188
Illegal NgoMIV site found at 3215
Illegal NgoMIV site found at 4986 - 1000COMPATIBLE WITH RFC[1000]
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