Part:BBa_K3002106
Level 1 – Mutant PETase + cCA + HA (Phytobrick)
This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), cCA secretion signal (BBa_K3002007), HA-tag (BBa_K3002017) and the coding sequence of the mutant PETase (BBa_K3002014) plus the MoClo connectors for positions B1-B2 (BBa_K3002302), B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).
Construct is secreted through secretion signal cCA. The secretion signal cCA shows minor secretion of the protein without combination with SP20 tag. Construct containing cCA leads to very low yield of the secreted protein in comparison to the other secretion signals.
Analysis of the secretion of MUT-PETase with secretion signals cCA, GLE, and ARS. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase equipped with secretion signals from carbonic anhydrase (cCA), gamete lytic enzyme (GLE) and arylsulfatase (ARS). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs shown in (a) 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.Transformants C12 and A27 introduced in Figures 4 and 5, respectively, served as positive controls. The white arrow points to the MUT-PETase.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1069
Illegal PstI site found at 2042 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1069
Illegal PstI site found at 2042 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1069
Illegal PstI site found at 2042 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1069
Illegal PstI site found at 2042
Illegal NgoMIV site found at 804
Illegal NgoMIV site found at 831
Illegal NgoMIV site found at 2482 - 1000COMPATIBLE WITH RFC[1000]
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