Difference between revisions of "Part:BBa K3128018:Design"

Latest revision as of 20:37, 17 October 2019

OmpX-WT protein fused with T25 subpart of Bordetella Pertussis AC under constitutive promoter

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1471
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE 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.

References

EUROMEDEX BACTH

@@ Line 7: / Line 7: @@
 ===Design Notes===
-Euromedex BACTH kit contains the pKNT25 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 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.
+https://static.igem.org/mediawiki/parts/thumb/7/79/T--Grenoble-Alpes--bba18.1.jpg/800px-T--Grenoble-Alpes--bba18.1.jpg.png<br>
+https://static.igem.org/mediawiki/parts/thumb/1/19/T--Grenoble-Alpes--bba18.2.jpg/800px-T--Grenoble-Alpes--bba18.2.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/a/ad/T--Grenoble-Alpes--bba18.3.jpg/800px-T--Grenoble-Alpes--bba18.3.jpg.png<br>
+<br>
+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===
-pKNT25 plasmid from Euromedex BACTH kit was used. OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
+T25 is from <i>bordetella pertussis</i><br>
+Ompx is from <i> Escherichia coli</i> <br>
+<i> pKT25 </i> plasmid from Euromedex BACTH kit was used (containing the T25 subpart and the leucine zipper gene).<br>
+OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
 ===References===
+[https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=2ahUKEwiw4qGr_ozlAhULA2MBHZNsBdUQFjACegQIBRAC&url=http%3A%2F%2Fstatic.bioport.cn%2Fdata%2Fupload%2Fproduct%2Fspecification%2F396%2F1342594983673_396560.pdf&usg=AOvVaw2TFscbzRiSzNjAvyMsZDHY EUROMEDEX BACTH]