Protocols
Protocols
Standard recombineering sites (grey triangles) can be used for recombination of different SEGA parts that we term SEGA "bricks". The recombination of these sites can be achieved using λ-red recombineering or any other functional recombineering system. The up- (uG) and downstream (uG) gadgets function as selection and counterselection markers for the exchange of parts.
Integration of a landing pad brick
A landing pad brick contains the full sized landing pad. This brick can be integrated by utilizing the downstream gadget tetA. In this case, tetA confers resistance to tetracycline and selection can be made on 50 µg/ml tetracycline
Integration of a control brick
A control brick contains either one or all control elements of the SEGA landing pad and can be used if the user wishes to exchange the existing control elements in the collection. This brick can be integrated using the upstream gadget galK. In this case, galK is used as a counterselection marker, since it makes the cells sensitive to 2-deoxy-galactose (DOG)
Integration of a cargo brick
A cargo brick contains any gene-of-interest that the user wants to integrate into the landing pad. This brick can be integrated using the donwstream gadget tetA. In this case, tetA functions as a counterselection marker, since it makes the cells sensitive to NiCl2
Integration of a controlled cargo brick
A controlled cargo brick contains both a new cargo and new control elements. To integrate this brick, both counterselection markers galK and tetA can be used. Selection can be achieved using 2-deoxy-galactose or NiCl2
Sequences of the recombineering sites that are used to integrate the different SEGA bricks
Site | Middle Gadget | Sequence (5'-3') | Comments |
RS1 | CCAAATTCCAGAAAAGAGACGCTTTCGAGCGTCTTTTTTCGTTTTGGTCC | Use for integration of control bricks/controlled cargo brick | |
RS2 | BCD | CAGTACGAAAATTGCTTTCATATATAGTTTCTCCTCTTTAATCTCTAGTA | Use for integration of control bricks |
RS2-cargo | BCD | ATTTTCGTACTGAAACATCTTAATCATGCTAAGGAGGTTTTCTAATG | Use for Integration of SEGA cargo bricks |
RS2-cargo | Ubiquitin | TGGAGCTGCAGAATGAAACCCAGTCAGCAACTTCTGACGCTTCTGGCGGT | Use for Integration of SEGA cargo bricks |
RS2-cargo | TEV C2:low* | TACTAGAGATTAAAGAGGAGAAACGGGGGATGGAGAACCTGTACTTCCAG | Use for Integration of SEGA cargo bricks |
RS2-cargo | TEV C2:medium* | TACTAGAGATTAAAGAGGAGAAAAACTACATGGAAAACCTGTACTTCCAG | Use for Integration of SEGA cargo bricks |
RS2-cargo | TEV C2:high* | TACTAGAGATTAAAGAGGAGAAAATATCGATGGAGAACCTGTACTTCCAG | Use for Integration of SEGA cargo bricks |
RS2-cargo | PROTi | CTGTACTTCCAGTTTCGTAGCAAAGGAGAAGAACTTGTCACTGGAGTT | Use for Integration of SEGA cargo bricks |
RS2-cargo | PelB | CGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC | Use for Integration of SEGA cargo bricks |
RS2-cargo | YebF | ATCAGGTGAGCGTCGATTGCAAAGCCGGCATGGCGGAGTACCAACGCCGC | Use for Integration of SEGA cargo bricks |
RS3 | CTAAGGATTTTTTTTATCTGGACAATTGTCTCAGGTCGAGGTGGCCCGGC | Use for integration of cargo bricks/controlled cargo brick | |
RS3-trunc | CTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGGCAGTAC | Use for truncation of tetA | |
RS3-recon | CGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCGCAAGAGGCCCGGCAGTAC | Use for reconstitution of tetA |
*In the case of the TEV-mGadget it is necessary to use homology sequences that are specific for each C2 site in order to create a scarless integration with the possibility to create a custom N-terminus on demand.
