Difference between revisions of "Part:BBa K1060011"
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<partinfo>BBa_K1060011 short</partinfo>
 
<partinfo>BBa_K1060011 short</partinfo>
  
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For the production of the alarmhormone,(E)-&#946;-farnesene, a sesquiterpene synthase gene (GenBank Accession No. AJ271793) was put behind a medium (constitutive) promoter and medium RBS (<partinfo>BBa_K608006</partinfo>). Additionally a Lac operator sequence (Gilbert & Maxam, 1973) was introduced to regulate its expression.
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For the production of the alarmhormone, (E)-&#946;-farnesene, a sesquiterpene synthase gene (GenBank Accession No. AJ271793) was put behind a medium (constitutive) promoter and medium RBS (<partinfo>BBa_K608006</partinfo>). Additionally a Lac operator sequence (Gilbert & Maxam, 1973) was introduced to regulate its expression.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1060011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1060011 SequenceAndFeatures</partinfo>
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https://static.igem.org/mediawiki/2013/b/b1/BBa_K10600011_gel_pic.jpg
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==SDS-PAGE confirmation==
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As another approach to prove our constructs (<partinfo>BBa_K1060009</partinfo>, <partinfo>BBa_K1060011 </partinfo> and <partinfo>BBa_K1060014 </partinfo>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/>
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Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.
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[[File:EBFS_protein.JPG|600px|center]]
  
  

Latest revision as of 01:42, 5 October 2013

Beta Farnesene production

For the production of the alarmhormone, (E)-β-farnesene, a sesquiterpene synthase gene (GenBank Accession No. AJ271793) was put behind a medium (constitutive) promoter and medium RBS (BBa_K608006). Additionally a Lac operator sequence (Gilbert & Maxam, 1973) was introduced to regulate its expression.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 209
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1409
    Illegal BamHI site found at 709
    Illegal XhoI site found at 671
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 281

BBa_K10600011_gel_pic.jpg


SDS-PAGE confirmation

As another approach to prove our constructs (BBa_K1060009, BBa_K1060011 and BBa_K1060014), we transformed our different EBF synthase bricks in an E.coli expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.

EBFS protein.JPG