Difference between revisions of "Part:BBa K3128018:Design"
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===Design Notes=== | ===Design Notes=== | ||
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| + | EcoRI and PstI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.<br> | ||
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Euromedex BACTH kit contains the pKT25 plasmid with a MCS cassette followed by the T25 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T25 subpart. To respect RFC rules, the plasmid has been amplified by PCR using a couple of primers that binds to the boundary sequences of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the gene of the protein of interest in the system. The plasmid was digested by the corresponding enzymes and the protein gene's was inserted. | Euromedex BACTH kit contains the pKT25 plasmid with a MCS cassette followed by the T25 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T25 subpart. To respect RFC rules, the plasmid has been amplified by PCR using a couple of primers that binds to the boundary sequences of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the gene of the protein of interest in the system. The plasmid was digested by the corresponding enzymes and the protein gene's was inserted. | ||
Latest revision as of 20:37, 17 October 2019
OmpX-WT protein fused with T25 subpart of Bordetella 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 1471
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
EcoRI and PstI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.
Euromedex BACTH kit contains the pKT25 plasmid with a MCS cassette followed by the T25 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T25 subpart. To respect RFC rules, the plasmid has been amplified by PCR using a couple of primers that binds to the boundary sequences of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the gene of the protein of interest in the system. The plasmid was digested by the corresponding enzymes and the protein gene's 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.