Part:BBa_K5121020:Design

rebB C-terminal LPETG

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 313
  

Design Notes

An LPETG peptide motif must be added to the C-terminus of rebB to make it compatible with Sortase A-mediated conjugations. The DNA sequence coding for the LPETG motif was incorporated using a two fragment PCR of the original reb1 plasmid (BBa_K5121011). Using primers with complementary overhangs, the sequence 5’-ctcccagaaacagga-3’ was added immediately adjacent to the stop codon (Figure 1). 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.

Figure 1. Plasmid map of the Reb1 plasmid including primer targeting sites that were used to add in the rebB C-terminal LPETG motif.

Source

E. coli