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Team:Groningen/parts fail
From 2012.igem.org
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| + | <td><z3>BBa_K592010</z3></td> | ||
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This BioBrick was intended to be expressed inside <i>E. coli</i> as described by <a class="z2link" href="http://partsregistry.org/Part:BBa_K592010">iGEM Uppsala-Sweden 2011</a>. In our project, we successfully expressed it inside <i>Bacillus subtilis</i> under regulation of sboA promoter. SboA-AmilGFP was shown to be very weakly expressed in <i>Bacillus subtilis</i> on LB plate without rotten meat induction (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 experiment (see our <a class="z2link" href="https://2012.igem.org/Team:Groningen/Sensor">sensor page</a>). First, we inoculated <i>B. subtilis</i>(SboA-AmilGFP) and <i>B. subtilis</i>(Wildtype) from plate into flasks of Luria Broth subjected to spoiled meat and without meat. 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><br> | This BioBrick was intended to be expressed inside <i>E. coli</i> as described by <a class="z2link" href="http://partsregistry.org/Part:BBa_K592010">iGEM Uppsala-Sweden 2011</a>. In our project, we successfully expressed it inside <i>Bacillus subtilis</i> under regulation of sboA promoter. SboA-AmilGFP was shown to be very weakly expressed in <i>Bacillus subtilis</i> on LB plate without rotten meat induction (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 experiment (see our <a class="z2link" href="https://2012.igem.org/Team:Groningen/Sensor">sensor page</a>). First, we inoculated <i>B. subtilis</i>(SboA-AmilGFP) and <i>B. subtilis</i>(Wildtype) from plate into flasks of Luria Broth subjected to spoiled meat and without meat. 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><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="385"></img><br><br> | <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="385"></img><br><br> | ||
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| - | + | Left: from left to right: Wildtype grown without meat, <i>B. subtilis</i>(SboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, B.s.(SboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat. | |
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| - | Left: from left to right: Wildtype grown without meat, <i>B. subtilis</i>(SboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, B.s.(SboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.< | + | </tr> |
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Right: Pelleted cells after 16 hour growth with/without spoiled meat | Right: Pelleted cells after 16 hour growth with/without spoiled meat | ||
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Revision as of 20:54, 26 September 2012
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This BioBrick was intended to be expressed inside E. coli as described by iGEM Uppsala-Sweden 2011. In our project, we successfully expressed it inside Bacillus subtilis under regulation of sboA promoter. SboA-AmilGFP was shown to be very weakly expressed in Bacillus subtilis on LB plate without rotten meat induction (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 experiment (see our sensor page). First, we inoculated B. subtilis(SboA-AmilGFP) and B. subtilis(Wildtype) 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. |
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| Left: from left to right: Wildtype grown without meat, B. subtilis(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 |
This is the list of incorrect BioBricks that we used from 2012 distribution kit.
| Name: | Gram-positive Shuttle Vector for Chromosomal Integration |
| Intended Purpose: | Plasmid backbone for cloning in Bacillus subtilis |
| Testing Protocol: | Restriction analysis (dual cut with EcoRI and PstI) |
| Results: | The restriction pattern showed that this biobrick is not as described in the parts registry. The cut plasmid is not a single band and shorter than the expected size. |
| Gel Pic |
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| Name: | Multi-host vector pTG262 converted to BioBrick vector |
| Intended Purpose: | Plasmid backbone for cloning in Bacillus subtilis |
| Testing Protocol: | Restriction analysis (single cut) and restriction analysis (dual cut) |
| Results: | The restriction enzymes cut at illegal site(s) |
| Gel Pic |
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| After discovering that we did not receive these BioBrick as described in the registry, we took initiative to make our own Bacillus subtilis backbone to accommodate our project's needs. These unexpected BioBricks cost us some time to reconstruct our backbone plan, but in the end we were able to come up with a new backbone plan, the BBa_K818000. |
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