Part:BBa_K3128029:Design
COMP fused with Leucine Zipper and T25 subpart of B.Pertussis AC under constitutive promoter
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1568
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 179
Design Notes
EcoRI and PstI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.
This biobrick has a TAG codon (Y121F' mutation) inside OmpX (amber TAG) sequene which can be used to introduce any Non Natural Amino acid outside the cell (more informations).
The signal peptide has a conformation enabling the protein addressing to the membrane.
This sequence is cut after the translocation.
If the COMP gene is used with the signal peptide, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein LeucineZipper-COMP.
Euromedex BACTH kit contains the pUT18 plasmid with a MCS cassette followed by the T18 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T18 subpart. To respect RFC rules, the plasmid has been amplificated by PCR using primers that bind from either site of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the protein in the system. The plasmid was digested by the corresponding enzymes and the protein gene was inserted.
Source
T25 is from bordetella pertussis
Ompx is from Escherichia coli
pKT25 plasmid from Euromedex BACTH kit was used (containing the T25 subpart and the leucine zipper gene).
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.