Difference between revisions of "Part:BBa K3128028:Design"
| Line 7: | Line 7: | ||
===Design Notes=== | ===Design Notes=== | ||
| + | https://static.igem.org/mediawiki/parts/thumb/3/3d/T--Grenoble-Alpes--bba28.1.jpg/800px-T--Grenoble-Alpes--bba28.1.jpg.png <br> | ||
| + | https://static.igem.org/mediawiki/parts/thumb/f/fa/T--Grenoble-Alpes--bba28.2.jpg/800px-T--Grenoble-Alpes--bba28.2.jpg.png <br> | ||
| + | EcoRI and PstI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.<br> | ||
| + | https://static.igem.org/mediawiki/parts/thumb/c/c8/T--Grenoble-Alpes--bba28.3.jpg/800px-T--Grenoble-Alpes--bba28.3.jpg.png <br> | ||
| + | |||
The signal peptide has a conformation enabling the protein addressing to the membrane.<br> | The signal peptide has a conformation enabling the protein addressing to the membrane.<br> | ||
This sequence is cut after the translocation.<br> | This sequence is cut after the translocation.<br> | ||
Revision as of 21:36, 17 October 2019
COMP fused with Leucine Zipper and T18 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 1436
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 977
Illegal NgoMIV site found at 1387
Illegal AgeI site found at 1193 - 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.
The signal peptide has a conformation enabling the protein addressing to the membrane.
This sequence is cut after the translocation.
If the OmpX gene is used without changes, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein leucine-zipper-OmpX.
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
T18 is from bordetella pertussis
Ompx is from Escherichia coli
pUT18 plasmid from Euromedex BACTH kit was used (containing the T18 subpart and the leucine zipper gene).
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.