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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> | | 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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| - | Parts characterization <br><br>
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| - | <b>Expression in ''<i>E. coli</i></b> <br> <br>
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| - | SboA-AmilGFP is strongly expressed in <i>E. coli</i>, 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" /></a><img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" /></a>
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| - | Left: Pellet of SboA-AmilGFP in <i>E. coli</i> dh5a. Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> dh5α. B3 is SboA-AmilGFP <br> <br>
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| - | '''Expression in <i>B. subtilis</i>''' <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. <br>
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| - | We (iGEM Groningen 2012) 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 (see our [https://2012.igem.org/Team:Groningen/Sensor| sensor page]).<br>
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| - | First, we inoculated <i>B. subtilis</i>(SboA-AmilGFP) and <i>B. subtilis</i>(Wildtype) from plate into flasks of Luria Broth:<br>
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| - | *Subjected to spoiled meat <br>
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| - | *Without meat <br> <br>
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| - | 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. <br>
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| - | [[Image:Groningen2012_ AP20120924_sboAamilGFPsetup_small.jpg|400px|]][[Image:Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg|400px|]] <br>
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| - | ''Left:from left to right: Wildtype grown without meat, B.s.(SboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, B.s.(SboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat. Right: Pelleted cells after 16 hour growth with/without spoiled meat.'' <br><br>
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| - | To check whether the difference in color was not the result of the promoter activation by the presence of meat in general, we also compared the growth of <i>B. subtilis</i>(SboA-AmilGFP) strain subjected to fresh meat and rotten meat. We grew the strain in Luria Broth in the microarray setup for 12 hours and measured OD (600 nm), Absorbance (395 nm) and assayed the color of the cells when pelleted. In the curves below you can see the results: while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster and in a larger amount when the device strain was subjected to volatiles from spoiling meat. <br>
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| - | [[Image:Groningen2012_RR_absorbance_vs_time.jpg]][[Image:Groningen2012_RR_growth_in_micarraysetup.png]]<br>
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| - | ''Left: Absorption of AmilGFP (395 nm) per amount of cells (OD(600)) of <i>Bacillus subtilis</i>(SboA-AmilGFP) strain grown for 12 hours while subjected to spoiled meat, fresh meat, or no meat. Right:Visibility of yellow color of pelleted cells by eye. Assay done with 5 previously made pellets of different color intensities as a reference to ensure objectivity.''<br>
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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.
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