Part:BBa_K3002220
L2 spectinomycin resistance + GLE_Mut-PETase_SP20HA
This composite part contains a spectinomycin resistance (BBa_K3002102) and the mutant PETase with the secretion signal GLE (BBa_K3002110) fused with an SP20HA-tag for easy detection via HA-antibody and enhanced secretion.
This part was used to investigate the secretion of the MUT-PETase alone without the MHETase during the screening process. As secretionsignal GLE was used and for detection the HA-tag in western blot analysis.
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
Illegal PstI site found at 3503
Illegal PstI site found at 4476 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2401
Illegal NheI site found at 2665
Illegal PstI site found at 3503
Illegal PstI site found at 4476 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2401
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2401
Illegal PstI site found at 3503
Illegal PstI site found at 4476 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2401
Illegal PstI site found at 3503
Illegal PstI site found at 4476
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 3238
Illegal NgoMIV site found at 3265
Illegal NgoMIV site found at 5036 - 1000COMPATIBLE WITH RFC[1000]
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