Difference between revisions of "Part:BBa K5121015:Design"

 
 
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===Design Notes===
 
===Design Notes===
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In order to maximise the chance of successful conjugation, the N-terminal cysteine was added onto the end of a glycine-serine linker, such that the full amino acid sequence added onto the N terminus was CGGGGS. This was achieved with a PCR of the original reb1 plasmid (BBa_K5121011) using overlapping primers to add in 5’-TGTGGTGGTGGTGGTTCT-3’ between the start codon and the rest of the rebB 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===
 
===Source===
  
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E. coli
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===References===
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Latest revision as of 12:26, 2 October 2024


rebB N-terminal CGGGGS


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
    COMPATIBLE WITH RFC[1000]


Design Notes

In order to maximise the chance of successful conjugation, the N-terminal cysteine was added onto the end of a glycine-serine linker, such that the full amino acid sequence added onto the N terminus was CGGGGS. This was achieved with a PCR of the original reb1 plasmid (BBa_K5121011) using overlapping primers to add in 5’-TGTGGTGGTGGTGGTTCT-3’ between the start codon and the rest of the rebB 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