Part:BBa_K3002113
Level 1 MHETase + cCA + HA
This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), the HA-tag (BBa_K3002017), the secretion signal cCA (BBa_K3002007) and the coding sequence of the MHETase (BBa_K3002037) plus the MoClo connectors for positions B1-B2 (BBa_K3002302), B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).
The MHETase in combination with the secretion signal cCA showed constant secretion by C.reinhardtii. This applies for the MHETase alone but also for the MHETase in combination with other level 1 constructs of the PETase. Constructs including cCA lead to a high yield of the secreted MHETase.
MUT-PETase destined for secretion gets stuck inside the cell. (a) Level 2 MoClo construct harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase genes. MUT-PETase and MHETase are equipped with the secretion signal from carbonic anhydrase (cCA). See Figure 1 for the description of other parts. (b) Seven days old cultures of transformants generated with the construct shown in (a) were centrifuged and proteins in the culture medium were precipitated by TCA and analysed by immunoblotting using an anti-HA antibody. The black arrow represents MHETase. (c) Whole-cell proteins of UVM4 cells transformed with construct L2C shown in (a) were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. (d) Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.
Quantification of secreted MHETase and MUT-PETase. (a) Transformants generated with constructs C, J, M, N, and O (Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Whole-cell extracts of strain B1-TIG-HA for which concentrations of the HA-tagged TIG protein are known are loaded next to the lyophilized supernatants. The black arrow points to MHETase, the white arrows to MUT-PETase. The supernatant of a culture with the UVM4 strain were loaded as negative control. (b) Maximum cell densities, doubling times, daily growth rates, yields of MHETase and PETase and daily productivity of both combined were calculated for the transformant lines indicated.
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 1327
Illegal PstI site found at 1651
Illegal PstI site found at 1994
Illegal PstI site found at 2804 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1327
Illegal PstI site found at 1651
Illegal PstI site found at 1994
Illegal PstI site found at 2804
Illegal NotI site found at 1662 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2572
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1327
Illegal PstI site found at 1651
Illegal PstI site found at 1994
Illegal PstI site found at 2804 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1327
Illegal PstI site found at 1651
Illegal PstI site found at 1994
Illegal PstI site found at 2804
Illegal NgoMIV site found at 1260
Illegal NgoMIV site found at 1721
Illegal NgoMIV site found at 1751
Illegal NgoMIV site found at 2066
Illegal NgoMIV site found at 2084
Illegal NgoMIV site found at 2120
Illegal NgoMIV site found at 2763 - 1000COMPATIBLE WITH RFC[1000]
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