Part:BBa_K5121014:Design
rebA C-terminal CGGGGS
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
In order to maximise the chance of successful conjugation, the C-terminal cysteine was added onto the end of a glycine-serine linker, such that the full amino acid sequence added onto the C-terminus was SGGGGC. This was achieved with a PCR of the original reb1 plasmid (BBa_K5121011) using overlapping primers to add in 5’-TCTGGTGGTGGTGGTTGT-3’ between the start codon and the rest of the rebA open reading frame. Rather than amplifying the whole plasmid in one reaction, the plasmid was amplified in two sections — each containing one of the two overlapping primers and a primer within the kanamycin resistance gene — followed by gibson assembly of the two fragments to reconstruct the full plasmid. This avoided annealing of the overlapping primers. The kanamycin resistance gene was chosen as the site of the second set of primers to ensure only cells with correctly assembled fragments at this site were viable — reducing the possible set of incorrectly assembled fragments.
Source
E. coli