Part:BBa_K1352010:Design

Ag43 autotransporter (ORF)

Assembly Compatibility:

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

  Illegal NgoMIV site found at 709
  Illegal NgoMIV site found at 1933
  Illegal NgoMIV site found at 2443
  Illegal NgoMIV site found at 2464
  Illegal AgeI site found at 1459
  Illegal AgeI site found at 2203
  Illegal AgeI site found at 2617
  Illegal AgeI site found at 2859
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

6 additional PstI restriction sites ( C_TGCA^G) observed within the open reading frame of Ag43 from BioBrick BBa_K759001 have been removed to comply with iGEM Assemly Standard RCF10. Synonymous mutations were introduced within the coding region of Ag43 through PCR-based Site-Directed Mutagenesis (Agilent Lightning Quik-Change mutagenesis kit) to destroy all 6 PstI sites. For restriction sites number 1,2,3,4 and 6, CTGCA^G has been converted to CTGCAA. For restriction site number 5, CTGCA^G has been changed to CTGCGG.

Figure 1. Gel electrophoresis on standard restriction digests of BBa_K759001 (top tier)versus BBa_K1352000 (bottom) Restriction digests were separated on a 1% agarose gel. Lane 1,10; NEB 1 kb size standards ladder, Lane 2,11; uncut plasmid, Lanes 3-6, and 12-15; plasmid cut with either EcoRI, XbaI, SpeI or PstI respectively, Lane 7 and 16; plasmid cut with EcoRI+PstI, Lane 8 and 17; plasmid cut with XbaI+SpeI, Lane 9 and 18; NEB 1 kb size standards ladder.

Media:Example.ogg

Source

PCR amplified from BioBrick BBa_K759001, then subjected to Site-Directed Mutagenesis.