Difference between revisions of "Part:BBa K1725350"
 
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<partinfo>BBa_K1725350 short</partinfo>
 
<partinfo>BBa_K1725350 short</partinfo>
  
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Part K1725350 is a <i>luxABG</i> gene assembly under the pBAD promoter generated by BioBrick assembly by combining <i>luxA</i> (<partinfo>K1725200</partinfo>), <i>luxB</i> (<partinfo>K1725201</partinfo>) and <i>luxG</i> (<partinfo>K1725205</partinfo>) ribosome binding site (RBS) libraries together. RBS <partinfo>K1725305</partinfo>, <partinfo>K1725309</partinfo> and <partinfo>K1725302</partinfo> are upstream of <i>luxA</i>, <i>luxB</i> and <i>luxG</i> genes, respectively. The assembly has been shown to induce high levels of bioluminescence in <i>E. coli</i> in a presence of decanal. [http://2015.igem.org/Team:Glasgow/Description More information] about the part.  
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Part K1725350 is a <i>luxABG</i> gene assembly under the pBAD promoter generated by BioBrick assembly of <i>luxA</i> (<partinfo>K1725200</partinfo>), <i>luxB</i> (<partinfo>K1725201</partinfo>) and <i>luxG</i> (<partinfo>K1725205</partinfo>) ribosome binding site (RBS) libraries. RBS <partinfo>K1725305</partinfo>, <partinfo>K1725309</partinfo> and <partinfo>K1725302</partinfo> are upstream of <i>luxA</i>, <i>luxB</i> and <i>luxG</i> genes, respectively. The assembly has been shown to induce high levels of bioluminescence in <i>E. coli</i> in a presence of decanal. [http://2015.igem.org/Team:Glasgow/Description More information] about the part.  
  
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==Design of RBS library for <i>luxABG</i>==
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==Design of the RBS library for <i>luxABG</i>==
 
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. Just for the <i>luxABG</i> assembly, there are over 32000 different variants.
 
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. Just for the <i>luxABG</i> assembly, there are over 32000 different variants.
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[[File:Streaks_of_luxABG_RBS.png|230px|thumb|upright|Streaks of <i>E. coli</i> colonies with <i>luxABG</i> RBS library. Top left colony is transformed with K1725350.]]
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[[File:Streaks_of_luxABG_RBS.png|280px|thumb|upright|Streaks of <i>E. coli</i> colonies with <i>luxABG</i> RBS library. Top left colony is transformed with K1725350.]]
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[[File:GlasgowTeam_RBSlibrarydesign.jpg|650px|thumb|none|Design of the RBS library for the <i>lux</i> operon ]]
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[[File:GlasgowTeam_RBSlibrarydesign.jpg|620px|thumb|none|Design of the RBS library for the <i>lux</i> operon ]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 23:51, 18 September 2015

pBAD/araC.luxABG - bright

Part K1725350 is a luxABG gene assembly under the pBAD promoter generated by BioBrick assembly of luxA (BBa_K1725200), luxB (BBa_K1725201) and luxG (BBa_K1725205) ribosome binding site (RBS) libraries. RBS BBa_K1725305, BBa_K1725309 and BBa_K1725302 are upstream of luxA, luxB and luxG genes, respectively. The assembly has been shown to induce high levels of bioluminescence in E. coli in a presence of decanal. [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. Just for the luxABG assembly, there are over 32000 different variants.

Streaks of E. coli colonies with luxABG RBS library. Top left colony is transformed with K1725350.
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
  • 21
    INCOMPATIBLE WITH RFC[21]
    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 SapI site found at 961