Part:BBa_K3002233
L2 spectinomycin resistance + MutateM_HA
This composite part contains a spectinomycin resistance (BBa_K3002102) and the Mutate M from iGEM16_TJUSLS_China (BBa_K3002101) fused with an HA-tag for easy detection via HA-antibody.
The constructs encodes the Mutate M PETase from iGEM16_TJUSLS_China (BBa_K3002101), it is expressed in the cytosol of C. reinhardtii. The PETase is essential for the degradation of PET into MHET. As selection marker an aadA cassette which mediates spectomycine resistance is used.
Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing the construct were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control.
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 2401
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2401
Illegal NheI site found at 2665 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2401
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2401
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2401
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 4138 - 1000COMPATIBLE WITH RFC[1000]
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