Difference between revisions of "Part:BBa K1060009"
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==SDS-PAGE confirmation==
 
==SDS-PAGE confirmation==
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As another approach to prove our constructs (<a href="https://parts.igem.org/Part:BBa_K1060009">BBa_K1060009</a>, <a href="https://parts.igem.org/Part:BBa_K1060011">BBa_K1060011</a> and <a href="https://parts.igem.org/Part:BBa_K1060014">BBa_K1060014</a>), 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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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/>
 
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.
 
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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https://static.igem.org/mediawiki/2013/7/76/EBFS_protein.JPG

Revision as of 00:34, 5 October 2013

Medium constitutive expression of EBF synthase from Artemisia annua

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).


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 177
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1368
    Illegal BamHI site found at 677
    Illegal XhoI site found at 639
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 249


Construct digestion

BBa_K1060009_gel_pic.jpg


System testing

Our pilot experiment with aphid indicated that our brick performed the desired function, for the video of aphids experiment, please go to [http://www.youtube.com/watch?v=n-pMp8-XdUo Aphid Repellence video].
In this video you will find our first behavioural experiment with aphids. An EBF-producing bacterium plate (E. coli BL21(DE3) on LB with 5mM Mg2+ and Chloramphenicol is placed on the left, a control plate on the right, aphids are placed on the leaf in the middle. The first results indicate a positive trend, as we can see them moving from left to right. (You may have to watch the video in full screen to clearly see the aphids.) For more information on the experimental setup and a discussion of the results go to [http://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBF#aphid%20experiments here].

For more infromation see the experience page or go to [http://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBF#aphid%20experiments here].


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