Difference between revisions of "Part:BBa K1725352"
(→‎Design of the RBS library for luxABG)
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==Design of the RBS library for <i>luxABG</i>==
 
==Design of the RBS library for <i>luxABG</i>==
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For the construction of the RBS library, a master sequence based on the RBS B0032 was used. 4 nucleotides within the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator. Originally, RBS Library was designed for every gene in luxABGCDE operon (<partinfo>K1725352</partinfo>) in order to optimise bioluminescence in E. coli. 32 different BioBrick RBS variants were incorporated upstream of each of the <i>lux</i> genes by PCR, and different libraries were then combined together by BioBrick assembly. This method could potentially produce over 1 billion different variants of the <i>lux</i> operon. We report that final construct K1725352 (pBAD/araC.luxABG.K1725080.luxCDE) is the brightest that we found out of 326 (over 1 billion) possible combinations.
+
For the construction of the RBS library, a master sequence based on the RBS B0032 was used. 4 nucleotides within the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator. Originally, RBS Library was designed for every gene in luxABGCDE operon (<partinfo>K1725352</partinfo>) in order to optimise bioluminescence in E. coli. 32 different BioBrick RBS variants were incorporated upstream of each of the <i>lux</i> genes by PCR, and different libraries were then combined together by BioBrick assembly. This method could potentially produce over 1 billion different variants of the <i>lux</i> operon. We report that the final construct K1725352 (pBAD/araC.luxABG.K1725080.luxCDE) is the brightest that we found out of over 1 billion possible combinations.
  
 
[[File:GlasgowTeam_RBSlibrarydesign.jpg|620px|thumb|none|Design of the RBS library for the <i>lux</i> operon ]]
 
[[File:GlasgowTeam_RBSlibrarydesign.jpg|620px|thumb|none|Design of the RBS library for the <i>lux</i> operon ]]

Revision as of 11:50, 19 September 2015


pBAD/araC.luxABG.R0011N.luxCDE - bright

Part K1725350 is a reorganised luxABGCDE operon with pBAD promoter upstream luxA and K1725080)promoter upstream luxC generated by BioBrick assembly of pBAD.luxABG-bright (BBa_K1725350)and K1725080.luxCDE-bright (BBa_K1725351).

[http://2015.igem.org/Team:Glasgow/Description More information] about the part.

Design of the RBS library for luxABG

For the construction of the RBS library, a master sequence based on the RBS B0032 was used. 4 nucleotides within the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator. Originally, RBS Library was designed for every gene in luxABGCDE operon (BBa_K1725352) in order to optimise bioluminescence in E. coli. 32 different BioBrick RBS variants were incorporated upstream of each of the lux genes by PCR, and different libraries were then combined together by BioBrick assembly. This method could potentially produce over 1 billion different variants of the lux operon. We report that the final construct K1725352 (pBAD/araC.luxABG.K1725080.luxCDE) is the brightest that we found out of over 1 billion possible combinations.

Design of the RBS library for the lux operon

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 1780
    Illegal NheI site found at 4057
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 6106
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 1608
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3156
    Illegal BsaI.rc site found at 5491
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 6659



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 1780
    Illegal NheI site found at 4057
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 6106
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 1608
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3156
    Illegal BsaI.rc site found at 5491
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 6659