Difference between revisions of "Part:BBa K615000:Design"

(→‎Design Notes)
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
−
HisB KO: 6 nucleotides (694-699) coding for amino acids V and E were deleted using MAGE.
+
*HisB KO: 6 nucleotides (694-699) coding for amino acids V and E were deleted using MAGE.
−
PyrF KO: Two early in-frame stop codons were made using MAGE to substitute 8T-->A and 16C-->G
+
*PyrF KO: Two early in-frame stop codons were made using MAGE to substitute 8T-->A and 16C-->G
−
rpoZ KO: Lambda red was used to replace gene with Zeocin resistance cassette.
+
*rpoZ KO: Lambda red was used to replace gene with Zeocin resistance cassette.
−
Kanamycin resistance cassette—Zif268 binding site and weak promoter—His3—URA3: inserted into 1529620 genomic locus using lambda red.
+
*Kanamycin resistance cassette—Zif268 binding site and weak promoter—His3—URA3: inserted into 1529620 genomic locus using lambda red.
−
MutS KO: replaced by chloramphenicol resistance cassette.
+
*MutS KO: replaced by chloramphenicol resistance cassette.
  
 
===Source===
 
===Source===

Revision as of 21:29, 21 September 2011

E. coli strain for His3-URA3 one-hybrid selection system


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 632
    Illegal PstI site found at 952
    Illegal PstI site found at 1762
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 632
    Illegal PstI site found at 952
    Illegal PstI site found at 1762
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 845
    Illegal BglII site found at 965
    Illegal BglII site found at 1478
    Illegal BglII site found at 1538
    Illegal BamHI site found at 1768
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 632
    Illegal PstI site found at 952
    Illegal PstI site found at 1762
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 632
    Illegal PstI site found at 952
    Illegal PstI site found at 1762
    Illegal NgoMIV site found at 181
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2458
    Illegal SapI site found at 121
    Illegal SapI site found at 331
    Illegal SapI.rc site found at 2305


Design Notes

  • HisB KO: 6 nucleotides (694-699) coding for amino acids V and E were deleted using MAGE.
  • PyrF KO: Two early in-frame stop codons were made using MAGE to substitute 8T-->A and 16C-->G
  • rpoZ KO: Lambda red was used to replace gene with Zeocin resistance cassette.
  • Kanamycin resistance cassette—Zif268 binding site and weak promoter—His3—URA3: inserted into 1529620 genomic locus using lambda red.
  • MutS KO: replaced by chloramphenicol resistance cassette.

Source

Base strain was EcNR2, a modified E. coli strain as described in Wang et al (2009).

URA3 and His3 are genes from yeast.

References: Harris H. Wang, Farren J. Isaacs, Peter A. Carr, Zachary Z. Sun, George Xu, Craig R. Forest, George M. Church. Programming cells by multiplex genome engineering and accelerated evolution. (2009). Nature, 460(7257):894-8.

References