Difference between revisions of "Part:BBa K1352010:Design"

 
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===Design Notes===
 
===Design Notes===
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6 additional PstI restriction sites ( C_TGCA^G) within the open reading frame of Ag43 from BioBrick BBa_K759001 have been removed through the insertion of synonymous mutations (CTGCAA). These were introduced through PCR-based Site-Directed Mutagenesis (Agilent Lightning Quik-Change mutagenesis kit)  
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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 Assembly 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.
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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.
  
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[[File:K1352000-restriction.jpg]]‎
  
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Figure 1. Gel electrophoresis on standard restriction digests of BBa_K759001 (top tier)versus BBa_K1352000 (bottom)
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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.
  
 
===Source===
 
===Source===

Latest revision as of 21:23, 2 October 2014


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 Assembly 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.

K1352000-restriction.jpg‎

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.

Source

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

References