Difference between revisions of "Part:BBa K1465401:Design"
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===Design Notes===
 
===Design Notes===
−
AB_alr_fwd (66 bp)
+
*Amplification of the pSB1C3-Backbone (<bbpart>BBa_J00450</bbpart>) using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_AB_fwd pSB1C3_AB_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_AB_rev pSB1C3_AB_rev]:<br>
−
5'-GAGTGGCAGGGCGGGGCGTAATTTTACTAGAAAGAGGAGAAACAAATGCAAGCGGCAACTGTTGTG-3'
+
  
−
AB_alr_rev (44 bp)
+
'''pSB1C3_AB_fwd (66 bp)'''<br>
−
5'-CTATCAACAGGAGTCCAAGCGAGCTTAATCCACGTATTTCATCG-3'
+
5'-CACAACAGTTGCCGCTTGCATTTGTTTCTCCTCTTTCTAGTAAAATTACGCCCCGCCCTGCCACTC-3'<br>
 +
'''pSB1C3_AB_rev (44 bp)'''<br>
 +
5'-CGATGAAATACGTGGATTAAGCTCGCTTGGACTCCTGTTGATAG-3'<br>
  
−
pSB1C3_AB_fwd (66 bp)
 
−
5'-CACAACAGTTGCCGCTTGCATTTGTTTCTCCTCTTTCTAGTAAAATTACGCCCCGCCCTGCCACTC-3'
 
  
−
pSB1C3_AB_rev (44 bp)
+
*Amplification of the alanine racemase (''alr'') from ''E. coli'' (<bbpart>BBa_K1172901</bbpart>) and the RBS (<bbpart>BBa_B0034</bbpart>) for the possibility of an antibiotic-free selection via complementation of the D-alanine auxotrophy. For the amplification the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#AB_alr_fwd AB_alr_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#AB_alr_rev AB_alr_rev] were used:<br>
−
5'-CGATGAAATACGTGGATTAAGCTCGCTTGGACTCCTGTTGATAG-3'
+
 
 +
'''AB_alr_fwd (66 bp)'''<br>
 +
5'-GAGTGGCAGGGCGGGGCGTAATTTTACTAGAAAGAGGAGAAACAAATGCAAGCGGCAACTGTTGTG-3'<br>
 +
'''AB_alr_rev (44 bp)'''<br>
 +
5'-CTATCAACAGGAGTCCAAGCGAGCTTAATCCACGTATTTCATCG-3'<br>
 +
 
 +
*[http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone containing also RFP  (<bbpart>BBa_J00450</bbpart>) as reporter and the alanine racemase  (<bbpart>BBa_K1172901</bbpart>) for the antibiotic-free selection- <br>
  
 
===Source===
 
===Source===
  
−
Gibson
+
<bbpart>BBa_J04450</bbpart>: RFP coding device.<br>
 +
<bbpart>BBa_K1172901</bbpart>: alanine racemase from ''E. coli''.<br>
  
 
===References===
 
===References===

Latest revision as of 21:47, 20 October 2014

Antibiotic-free selection plasmid (pSB1C3_alr_RFP)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3142
    Illegal NheI site found at 1893
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 3148
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3142
    Illegal BglII site found at 1969
    Illegal BamHI site found at 1267
    Illegal XhoI site found at 1033
    Illegal XhoI site found at 3018
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 3142
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 3142
    Plasmid lacks a suffix.
    Illegal XbaI site found at 3157
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 1551
    Illegal AgeI site found at 1851
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2094


Design Notes

  • Amplification of the pSB1C3-Backbone (BBa_J00450) using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_AB_fwd pSB1C3_AB_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_AB_rev pSB1C3_AB_rev]:
pSB1C3_AB_fwd (66 bp)

5'-CACAACAGTTGCCGCTTGCATTTGTTTCTCCTCTTTCTAGTAAAATTACGCCCCGCCCTGCCACTC-3'

pSB1C3_AB_rev (44 bp)

5'-CGATGAAATACGTGGATTAAGCTCGCTTGGACTCCTGTTGATAG-3'


  • Amplification of the alanine racemase (alr) from E. coli (BBa_K1172901) and the RBS (BBa_B0034) for the possibility of an antibiotic-free selection via complementation of the D-alanine auxotrophy. For the amplification the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#AB_alr_fwd AB_alr_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#AB_alr_rev AB_alr_rev] were used:
AB_alr_fwd (66 bp)

5'-GAGTGGCAGGGCGGGGCGTAATTTTACTAGAAAGAGGAGAAACAAATGCAAGCGGCAACTGTTGTG-3'

AB_alr_rev (44 bp)

5'-CTATCAACAGGAGTCCAAGCGAGCTTAATCCACGTATTTCATCG-3'
  • [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone containing also RFP (BBa_J00450) as reporter and the alanine racemase (BBa_K1172901) for the antibiotic-free selection-

Source

BBa_J04450: RFP coding device.
BBa_K1172901: alanine racemase from E. coli.

References