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	<z3><i>Update! (26th October 2012)</i></z3><br><br>		<z3><i>Update! (26th October 2012)</i></z3><br><br>
	After the European regional jamboree, we were back in the lab to build our planned constructs in the <A HREF="https://2012.igem.org/Team:Groningen/in_development"><FONT COLOR=#ff6700>development page</FONT></A>. We were able to construct the AmilGFP under regulation of P<i>wap</i>A, one of the rotten meat down-regulated promoter detected in microarray experiment.<br><br>		After the European regional jamboree, we were back in the lab to build our planned constructs in the <A HREF="https://2012.igem.org/Team:Groningen/in_development"><FONT COLOR=#ff6700>development page</FONT></A>. We were able to construct the AmilGFP under regulation of P<i>wap</i>A, one of the rotten meat down-regulated promoter detected in microarray experiment.<br><br>
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Revision as of 13:24, 26 October 2012

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.

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.


Update! (26th October 2012)

After the European regional jamboree, we were back in the lab to build our planned constructs in the development page. We were able to construct the AmilGFP under regulation of PwapA, one of the rotten meat down-regulated promoter detected in microarray experiment.

We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, PsboA, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of PwapA in the presence of fresh meat but not in the rotten meat and its amilGFP production in fresh meat is higher than leak productionof AmilGFP under regulation of PsboA in the same situation.

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.

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.

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 B. subtilis 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. 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.

Left: Absorption of AmilGFP (395 nm) per amount of cells (OD(600)) of Bacillus subtilis 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.

AmilGFP and AmilCP both are fluorescent proteins. We decided to quantify the amount of AmilGFP inside our Bacillus subtilis strain when subjected to spoiled meat and without meat. As a positive control, we paired the AmilGFP coding gene to the strong Bacillus subtilis promoter rrnB. We measured the fluorescence, the OD and color of the pellet of all four test subjects during growth for 12 hours. The picture above shows the difference in fluorescence after twelve hours. It is clear that in the presence of volatiles that produced by the spoiled meat, the sboA promoter was highly upregulated, thus more amilGFP was expressed. Previous tests showed that the intensity of AmilGFP expressed by Bacillus subtilis sboA-AmilGFP strain that was exposed to fresh meat was the same as the intensity of AmilGFP that was expressed by Bacillus subtilis sboA-AmilGFP strain exposed to a no-meat environment.

SboA-AmilCP

AmilCP is expressed less strongly in Bacillus subtilis than AmilGFP. On plate, not induced by volatiles, a faint blue-greyish color is visible after 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles. However, after placing Bacillus subtilis in our sticker and exposing the sticker to rotten meat volatiles, it turned into a clear purple color. See the sticker page for more information.