Team:Groningen/Construct

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<z1>Construct</z1>
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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.
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When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the
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production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced
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when the promoter is activated.
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Hover your mouse over the image to see a bigger version!
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<identificationP ><A HREF="https://2012.igem.org/Team:Groningen/identifyingPBADmeat" TARGET="_blank"><FONT COLOR=#ffffff>identification</FONT></A></identificationP>
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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
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and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside
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<i>E. coli</i>.
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<z2>Update! (26th October 2012)</z2>
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After the European regional jamboree, we were back in the lab to build our planned constructs in the
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<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>.
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                }
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We coupled  P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene.
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PigmentsP {
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We engineered the construct inside psac-cm backbone (BBa_K818000)
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                .Pigments1 {
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<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350">
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AmilGFP under regulation of P<i>wap</i>A
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The pigment production activity of P<i>wap</i>A-amilGFP  was  compared with the production of the pigment regulated by the up-regulated promoter
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(P<i>sbo</i>A) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of P<i>wap</i>A in the presence of
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fresh meat but absent in the presence of  rotten meat.
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<z1>Characterization</z1>
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<z2>SboA-AmilGFP</z2>
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<div class="Pigments2">
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<br>
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<PigmentsP ><A HREF="https://2012.igem.org/Team:Groningen/Pigments" TARGET="_blank"><FONT COLOR=#ffffff>pigment</FONT></A></Pigments>
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<z3>Expression in <i>E. coli</i></z3>
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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,  
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the yellow color is less visible compared to the cell pellet in liquid culture.
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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">
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<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400">
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<p class="caption">
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ConstructP {
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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> DH5a. B3 is SboA-AmilGFP.
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</p>
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                .Construct1 {
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<z3>Expression in <i>B. subtilis</i></z3>
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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).  
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This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to
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volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and
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<i>B. subtilis</i>Wildtype from plate into flasks of  Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>.  
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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:
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                        color: #FFF;
+
<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
-
                .Construct2 {
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yellow color at all.
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<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325">
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<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400">
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<p class="caption">
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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, <i>B.subtilis</i>(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.<br>
-
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(right) Pelleted cells after 16 hour growth with/without spoiled meat.
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<div class="Construct2">
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<br>
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<div class="Construct1">
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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
-
<ConstructP ><A HREF="https://2012.igem.org/Team:Groningen/Construct" TARGET="_blank"><FONT COLOR=#ffffff>construct</FONT></A></ConstructP>
+
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
-
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+
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:
-
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+
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster
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and in a larger amount when the device strain was subjected to volatiles from spoiling meat.
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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. <br>
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(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.
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<z5>AmilGFP</z5> and <z5>AmilCP</z5> both are <z5>fluorescent proteins</z5>. We decided to quantify the amount of AmilGFP inside our <i>Bacillus subtilis</i>
-
                .Death1 {
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strain when subjected to spoiled meat and without meat. As a positive control, we paired the AmilGFP coding gene to the <z5>strong <i>Bacillus subtilis</i>
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+
promoter rrnB</z5>. We measured the fluorescence, the OD and color of the pellet of all four test subjects during growth for 12 hours. The picture above
-
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+
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
-
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+
was highly upregulated, thus more amilGFP was expressed.
-
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Previous tests showed that the intensity of AmilGFP expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain that was exposed to fresh meat was the same as
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the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.
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<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 />
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<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" class="preview" width=700 height=450 />
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Hover your mouse over the image to see a bigger version!<br>
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<i>Bacillus subtilis</i>, 1000x, AmilGFP fluorescence measurement, exposure time = 50 ms, ex = 470 nm, em = 514 nm. Clockwise, from the top (left) 1) positive control: strong promoter rrnB with AmilGFP. 2) SboA-AmilGFP exposed to spoiled meat. 3)Wild type 4)SboA-AmilGFP grown without meat.
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<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 />
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150" height="150">
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Hover your mouse over the image to see a bigger version!<br>
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Color of pellets of sboA-GFP in a no-meat environment (above) and exposed to spoiled meat (below)after 6 hours(6H), 8 hours (8H), 10 hours (10H), and 12 hours (12H).
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</p>
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<div style="position:absolute; left: 20px; top: 560px;">
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<img src="https://static.igem.org/mediawiki/2012/c/c2/Groningen2012_RR_20120909_rotting.png" width="100" height="100">
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<br>
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<z2>SboA-AmilCP</z2>
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<div style="position:absolute; right: 0px; top: 550px;opacity:0.4; filter:alpha(opacity=40); z-index:-1;">
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<br>
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<img src="https://static.igem.org/mediawiki/2012/b/b6/Groningen2012_AD_20120802_BigCog.png" width="150" height="150">
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AmilCP is expressed less strongly in <i>Bacillus subtilis</i> than AmilGFP. On plate, not induced by volatiles, a faint blue-greyish color is visible after
-
</div>
+
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.
-
 
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However, after placing <i>Bacillus subtilis</i> in our sticker and exposing the sticker to rotten meat volatiles, it turned into a clear purple color.
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<div style="position:absolute; right: 30px; top: 575px;">
+
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.
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<img src="https://static.igem.org/mediawiki/2012/e/e7/Groningen2012_RR20120909_capsule.png" width="100" height="100">
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<p class="marginleft">
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<FONT COLOR=#ff6700><b>The standard construct is a combination of biobricks.</b><FONT>
+
<br>
<br>
-
 
-
<FONT COLOR=#ffffff SIZE=3pt style="margin-right=100px; margin-left=100px;"><ol>
 
-
<li>Backbone:Available in registry: BBa_K090403 (Backbone) - Single copy shuttle vector. Other option: build our own well-characterized backbone for our chassis.</li>
 
-
 
-
<li>BBa_K116632+BBa_K116602 (Positive feedback loop) - Uses the pRe promoter regulated by C11 and terminated by B0010. Other options: BBa_J37019;BBa_K116639; BBaK116609.</li>
 
-
 
-
 
-
<li>BBa_K274100 (Reporter) - Modified carotenoid pathway to produce red pigment.</li>
 
<br>
<br>
<br>
<br>
 +
<z2>Update! (26th October 2012)</z2>
<br>
<br>
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<br>
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<br>
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<br>
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<z3>Quantification of P<i>sbo</i>A expression by flow cytometry</z3>
-
<br>
+
<br><br>
-
<br>
+
To further characterize the difference in amilGFP expression under the P<i>sbo</i>A promoter, we measured the fluorescence of amilGFP (ex = 470 nm, em = 514 nm) by flow cytometry. We let our strain grow in the presence of spoiled and fresh meat for nine hours. As showed in the figures below, a clear difference in fluorescence can be seen after six hours of incubation.
-
<br>
+
We observed that the fluorescence intensity is also slightly influenced by the growth speed of the bacterium: the slower growing culture “fresh 2” (see pictures B and C) has a lower expression of amilGFP compared to the faster growing culture “fresh 1”. However, this difference can be neglected when compared to the difference of the cultures subjected to fresh and spoiled meat while having the same growth speed. This confirmed the importance of finding ways to control the growth of the bacterium inside our sticker by <a class="inlink" href="https://2012.igem.org/Team:Groningen/Modeling">modeling</a>.<br><br>
-
<br>
+
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<br>
+
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<li>Or, using BBa_K274002 (Reporter) - Violet pigment producer.</li>  
+
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<img src="https://static.igem.org/mediawiki/2012/a/a3/EJ_Groningen2012_RR-pretty_graphs.png" width=700 />
 +
<img src="https://static.igem.org/mediawiki/2012/a/a3/EJ_Groningen2012_RR-pretty_graphs.png" class="preview" width=1200 />
 +
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 +
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 +
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 +
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 +
<p class="caption">
 +
Hover your mouse over the image to see a bigger version!<br>
 +
Flow cytometry experiment: Bacillus subtilis with P<i>sbo</i>A-amilGFP (pre-cultured O/N and diluted to start OD(600)=0.1) was grown at 37 degrees Celsius for 9 hours while subjected to spoiled meat or fresh meat (on ice). Fluorescence was checked by flow cytometry every hour.
 +
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 +
<img src="https://static.igem.org/mediawiki/2012/f/f8/EJ_Groningen2012_RR-intensity_over_time.PNG" width=350 />
 +
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 +
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 +
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 +
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 +
<img src="https://static.igem.org/mediawiki/2012/0/0c/EJ_Groningen2012_RR-OD_measurement.PNG" width=321 />
 +
<img src="https://static.igem.org/mediawiki/2012/0/0c/EJ_Groningen2012_RR-OD_measurement.PNG" class="preview" width=521 />
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 +
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 +
Hover your mouse over the image to see a bigger version!<br>
 +
<b>(Left)</b>: Mean fluorescence intensity of amilGFP determined by flow cytometry, analyzed with WinMDI viewer (freeware) over time (see figure above). The expression of PsboA influenced by spoiled meat can differ significantly (spoiled 1 and 2), probably due to the high individual differences in meat spoilage per meat sample. The expression is significantly higher compared to the expression influenced by fresh meat. <b>(Right)</b> OD(600) of the cell cultures depicted in the left graph.
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{{Template:SponsorsGroningen2012}}
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<a href="https://2012.igem.org/Team:Groningen/Kill_Switch">
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+
<div style="position:absolute; right: 10px; bottom: 700px;">
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== Construct ==
 
-
The standard construct is a combination of biobricks.
 
-
*Backbone:Available in registry: BBa_K090403 (Backbone) - Single copy shuttle vector. Other option: build our own well-characterized backbone for our chassis.
 
-
* BBa_K116632+BBa_K116602 (Positive feedback loop) - Uses the pRe promoter regulated by C11 and terminated by B0010.  Other options: BBa_J37019;BBa_K116639; BBaK116609.
 
-
* BBa_K274100 (Reporter) - Modified carotenoid pathway to produce red pigment.
 
-
[[File:Groningen2012_JP_20120618_pRe-CII-Lycopene_Construct_A.png|680px|center]]
 
-
* Or, using BBa_K274002 (Reporter) - Violet pigment producer
 
-
[[File:Groningen2012_JP_20120618_pRe-CII-Violacein_Construct_A.png|680px|center]]
 

Latest revision as of 01:08, 27 October 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.

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 coupled PwapA, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. We engineered the construct inside psac-cm backbone (BBa_K818000)

AmilGFP under regulation of PwapA


The pigment production activity of PwapA-amilGFP was compared with the production of the pigment regulated by the up-regulated promoter (PsboA) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of PwapA in the presence of fresh meat but absent in the presence of rotten meat.

Characterization


SboA-AmilGFP

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 color 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 DH5a. B3 is SboA-AmilGFP.


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) 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) 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.

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Bacillus subtilis, 1000x, AmilGFP fluorescence measurement, exposure time = 50 ms, ex = 470 nm, em = 514 nm. Clockwise, from the top (left) 1) positive control: strong promoter rrnB with AmilGFP. 2) SboA-AmilGFP exposed to spoiled meat. 3)Wild type 4)SboA-AmilGFP grown without meat.

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Color of pellets of sboA-GFP in a no-meat environment (above) and exposed to spoiled meat (below)after 6 hours(6H), 8 hours (8H), 10 hours (10H), and 12 hours (12H).


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.


Update! (26th October 2012)

Quantification of PsboA expression by flow cytometry

To further characterize the difference in amilGFP expression under the PsboA promoter, we measured the fluorescence of amilGFP (ex = 470 nm, em = 514 nm) by flow cytometry. We let our strain grow in the presence of spoiled and fresh meat for nine hours. As showed in the figures below, a clear difference in fluorescence can be seen after six hours of incubation. We observed that the fluorescence intensity is also slightly influenced by the growth speed of the bacterium: the slower growing culture “fresh 2” (see pictures B and C) has a lower expression of amilGFP compared to the faster growing culture “fresh 1”. However, this difference can be neglected when compared to the difference of the cultures subjected to fresh and spoiled meat while having the same growth speed. This confirmed the importance of finding ways to control the growth of the bacterium inside our sticker by modeling.

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Flow cytometry experiment: Bacillus subtilis with PsboA-amilGFP (pre-cultured O/N and diluted to start OD(600)=0.1) was grown at 37 degrees Celsius for 9 hours while subjected to spoiled meat or fresh meat (on ice). Fluorescence was checked by flow cytometry every hour.



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(Left): Mean fluorescence intensity of amilGFP determined by flow cytometry, analyzed with WinMDI viewer (freeware) over time (see figure above). The expression of PsboA influenced by spoiled meat can differ significantly (spoiled 1 and 2), probably due to the high individual differences in meat spoilage per meat sample. The expression is significantly higher compared to the expression influenced by fresh meat. (Right) OD(600) of the cell cultures depicted in the left graph.





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