Difference between revisions of "Part:BBa K3128020:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | + | https://static.igem.org/mediawiki/parts/thumb/8/82/T--Grenoble-Alpes--BBa20.jpg/800px-T--Grenoble-Alpes--BBa20.jpg.png <br> | |
| − | + | https://static.igem.org/mediawiki/parts/thumb/b/bc/T--Grenoble-Alpes--BBa202.jpg/800px-T--Grenoble-Alpes--BBa202.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/9/9b/T--Grenoble-Alpes--BBa203.jpg/800px-T--Grenoble-Alpes--BBa203.jpg.png <br> | ||
| + | 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 ([https://2019.igem.org/Team:Grenoble-Alpes more informations]).<br> | ||
| + | <br> | ||
| + | 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. | ||
===Source=== | ===Source=== | ||
| − | pKNT25 plasmid from Euromedex BACTH kit was used. | + | T25 is from <i>bordetella pertussis</i><br> |
| + | Ompx is from <i> Escherichia coli</i> <br> | ||
| + | <i> pKNT25 </i> plasmid from Euromedex BACTH kit was used.<br> | ||
| + | COMP gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates. | ||
===References=== | ===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] | ||
Latest revision as of 20:21, 17 October 2019
COMP 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 1432
- 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.
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).
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.
Source
T25 is from bordetella pertussis
Ompx is from Escherichia coli
pKNT25 plasmid from Euromedex BACTH kit was used.
COMP gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.