Team:Groningen/Construct

From 2012.igem.org

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We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <i>E. coli</i>. <br><br>
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We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <i>E. coli</i>. <br><br><br>
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<z1>Characterization</z1>
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<z2>SboA-AmilGFP</z2>
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1.) <z3>Expression in <i>E. coli</i></z3><br>
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<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, the yellow colour is less visible compared to the cell pellet in liquid culture.<br>
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<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img>  <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img>
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<br>
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<p class=caption><i>
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Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br>
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Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i>
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2) <z3>Expression in <i>B. subtilis</i></z3>
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<br><br>
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sboA-AmilGFP was shown to be very weakly expressed in <i>Bacillus subtilis</i> on LB plate (faint color formation after 2 days). This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and <i>B. subtilis</i>Wildtype from plate into flasks of  Luria Broth subjected to <z4>spoiled meat</z4> and <z4>without meat</z4>. We grew <i>B. subtilis</i> containing sboA-AmilGFP device in the setup overnight (16 hours) at 37 degrees Celsius. In the picture below, you can see the result: <i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), while the same strain that was grown without meat only showed very faint yellow color. Both <i>B. subtilis </i> wildtype in this setup did not express yellow color at all.<br><br>
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<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"></img> <img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"></img>
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<p class=caption><i>Left picture, from left to right: Wildtype grown without meat, <i>B.subtilis</i>(sboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, <i>B.subtilis</i>(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.<br>
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Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p>
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Revision as of 22:15, 26 September 2012





Construct


Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When Bacillus subtilis senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.

  • Hover your mouse over the image to see a bigger version!


We use our Bacillus subtilis backbone (BBa_K818000) that has sacA and a chloramphenicol resistance gene for chromosomal integration and antibiotic screening of transformants respectively. This backbone also has E. coli origin of replication, so it can be amplified inside E. coli.


Characterization

SboA-AmilGFP

1.) Expression in E. coli
SboA-AmilGFP is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, the yellow colour is less visible compared to the cell pellet in liquid culture.


Left: Pellet of SboA-AmilGFP in E. coli DH5a.
Right: Plate with SboA connected to several pigment genes inside E. coli DH5α. B3 is SboA-AmilGFP.



2) Expression in B. subtilis

sboA-AmilGFP was shown to be very weakly expressed in Bacillus subtilis on LB plate (faint color formation after 2 days). This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in B. subtilis subjected to volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated B. subtilisSboA-AmilGFP and B. subtilisWildtype from plate into flasks of Luria Broth subjected to spoiled meat and without meat. We grew B. subtilis containing sboA-AmilGFP device in the setup overnight (16 hours) at 37 degrees Celsius. In the picture below, you can see the result: B. subtilis sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), while the same strain that was grown without meat only showed very faint yellow color. Both B. subtilis wildtype in this setup did not express yellow color at all.

Left picture, from left to right: Wildtype grown without meat, B.subtilis(sboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, B.subtilis(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat.

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