Team:Groningen/Construct

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<z1>Construct</z1>
 
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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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<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 />
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Hover your mouse over the image to see a bigger version!
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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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<br>
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Our construct idea is simple and effective: there will be a production of pigment under the regulation of rotten-meat reactive promoter. When the <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the production of downstream genes. We put pigment genes available downstream of the promoter so that the pigment would be produced when the promoter is activated.<br>
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After the European regional jamboree, we were back in the lab to build our planned constructs in the
-
</p>
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<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>.
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<div  align="center">
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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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<ul class="hoverbox">
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We engineered the construct inside psac-cm backbone (BBa_K818000)
-
<li>
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<br>
-
<a href="#"><img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /></a>
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<br>
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</p>
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<table class="centertable">
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<tr>
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<td align="center">
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<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350">
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</td>
 +
</tr>
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<tr>
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<td align="center">
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<p class="captionnomargin">
 +
AmilGFP under regulation of P<i>wap</i>A
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</p>
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</td>
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</tr>
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</table>
 +
<br>
 +
<p>
 +
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
 +
(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
 +
fresh meat but absent in the presence of  rotten meat.
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</p>
 +
<div class="cte2">
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<div class="ctd2">
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<z1>Characterization</z1>
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</div>
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</div>
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<p>
 +
<br>
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<z2>SboA-AmilGFP</z2>
 +
<br>
 +
<br>
 +
<z3>Expression in <i>E. coli</i></z3>
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<br>
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<br>
 +
<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.
 +
<br>
 +
</p>
 +
<table class="centertable">
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<tr>
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<td align="center">
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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 src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400">
 +
</td>
 +
</tr>
 +
</table>
 +
<p class="caption">
 +
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br>
 +
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.
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</p>
 +
<p>
 +
<br>
 +
<z3>Expression in <i>B. subtilis</i></z3>
 +
<br>
 +
<br>
 +
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 <z5>spoiled meat</z5> and <z5>without meat</z5>.
 +
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.
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<br>
 +
<br>
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</p>
 +
<table class="centertable">
 +
<tr>
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<td align="center">
 +
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325">
 +
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400">
 +
</td>
 +
</tr>
 +
</table>
 +
<p class="caption">
 +
(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>
 +
(right) Pelleted cells after 16 hour growth with/without spoiled meat.
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</p>
 +
<p>
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<br>
 +
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. 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.
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<br>
 +
<br>
 +
</p>
 +
<table class="centertable">
 +
<tr>
 +
<td align="center">
 +
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375">
 +
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315">
 +
</td>
 +
</tr>
 +
</table>
 +
<p class="caption">
 +
(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>
 +
(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.
 +
</p>
 +
<br>
 +
<p>
 +
<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>
 +
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>
 +
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
 +
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 <i>Bacillus subtilis</i> sboA-AmilGFP strain that was exposed to fresh meat was the same as
 +
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.
 +
<br>
 +
</p>
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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 />
 +
<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 />
 +
</a>
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</li>
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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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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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<z2>SboA-AmilCP</z2>
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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
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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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See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.
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We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and chloramphenicol resistance gene for chromosomal integration and transformants antibiotic screening. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <i>E. coli</i>.
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<z2>Update! (26th October 2012)</z2>
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<z3>Quantification of P<i>sbo</i>A expression by flow cytometry</z3>
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<br><br>
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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.
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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>
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There are several ideas to improve our current construct, such as: multi-colored pigment system and fine tuning of the pigment production.
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<z2>Tuning system of Pigment Production</z2>
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Hover your mouse over the image to see a bigger version!<br>
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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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The core concept behind the Food Warden is that it should pave the way to a more comprehensive, scientifically informed prediction of food edibility that goes beyond conventional best-before dates. The Food Warden as it is now is only a proof of principle. The goal is then to produce a system that is truly more accurate and reliable than the best-before date. The tuning needed requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br>
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<font color=#FF6700><b>1. </b></font>Volatile concentration: Building upon our gas chromatography approach in order to quantitatively assess the volatile production of spoiling meat.<br>
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<font color=#FF6700><b>2. </b></font>Degree of spoilage heath risk: The unrefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"> https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative</a>) makes this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of that standard for specific types of meat.<br>
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<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcomes of the previous two aspects of the tuning procedure. Once the relationship between volatile composition/concentration and health risk is elucidated to some degree, the pigment production can be tuned to fit this parameter.
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The pigment production can be tuned to a desired speed and sensitivity with different regulating promoter, different rbs, and with positive feedback system to increase the pigment production. One example of the positive feedback system that can be applied to increase pigment production under the regulation of the rotten meat promoter:<br></p>
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ad/Groningen2012_EJ_20120924_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/a/ad/Groningen2012_EJ_20120924_positive_feedback_construct.png" class="preview" width=700 /></a>
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When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is then formed so that the pigment and the inducer will be in the loop, increasing the production rate of the pigment. This system is meant to increase the production speed of the pigment. One of the possible set of inducible promoter-inducer is pRE promoter (BBa_K116603) with CII (BBa_K116602).
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<z2>Multi-colored Pigment System</z2>
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Hover your mouse over the image to see a bigger version!<br>
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<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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The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this in terms of user-friendliness that need to be addressed. The Food Warden can only do its job if it can grow properly upon breaking of the inner compartment of the sticker. It is plausible that manufacturing errors during the production of an eventual Food Warden product could lead to issues with the germination of the spores, resulting in a sticker that does not do its job. Eventualities include:<br>
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<font color=#FF6700><b>1. </b></font>Incorrect medium: In the event that the medium supplied in the sticker is not of the correct composition, the Food Warden will not grow, and therefore will not be able to identify spoiling meat.
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<font color=#FF6700><b>2. </b></font>Absence of viable spores: A defect sticker could be accidentally produced that either lacks spores capable of germination or lacks spores entirely.
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<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that
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If the user is not aware of these problems he or she may assume that the lack of pigment production simply means the meat in question is not spoiling yet, whereas it may already be spoiling and indeed will without any warning.
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Although these eventualities are a production process concern, it is our job to produce a system that minimizes the risk of these problems affecting the user. Therefore, we would have liked to build in a positive control that allows the user to confirm that the Food Warden in their sticker germinates and grows.  
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The positive control considered was the inclusion of a constitutively produced second pigment. This pigment serves as a signal to the user that the Food Warden is functional upon germination. This positive control pigment should of course not interfere with the visual detection of the warning pigment. To ensure this, the following option were considered:<br>
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<font color=#FF6700><b>1. </b></font>Choosing the two pigment colors such that the warning pigment color is highly dominant over the control pigment color.<br>
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<font color=#FF6700><b>2. </b></font>Placing the pigment under the control of a weak constitutive promoter, producing the control pigment at minimum levels required for user detection, therefore more easily being overpowered by the warning pigment.<br>
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<font color=#FF6700><b>3. </b></font>Placing the pigment under the control of a constitutive promoter, identified in our microarray analysis, that down-regulates the gene it controls under spoiling meat conditions, allowing the warning pigment to more easily overpower the control pigment. <font color=#FF6700><b>[[[diagram listing a few down regulated promoters we identified that we could use]]]</b></font><br>
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<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows for repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br>
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The construct can be put as following:<br>
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<img class="centerimage" src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width="600">
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<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to specific compound to change its color. The control pigment is under the regulation of a constitutive promoter while the rotten meat promoter regulates the compound needed to change the control pigment's color.<br>
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The construct can be put as following:<br><br>
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<img class="centerimage" src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width="600">
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When the rotten meat is activated, the compound needed to change the control pigment's color and the repressor will be produced. The control pigment production will be stopped and the existing control pigment will start to react to the compound, changing its color and becoming the warning pigment. This can be achieved using existing biobrick: BBa_K274100 (lycopene, red pigment) and adding the compound (CrtY) encoded by BBa_K118008 to make the red color from lycopene into yellow color (beta-carotene). Another construct using positive feedback loop for the warning-pigment-compound without stopping the control pigment production is as following:<br><br>
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<img class="centerimage" src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width="600">
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In this system, the control pigment will still be produced even when the rotten meat promoter activates. The production speed of the second pigment compound which reacts to the control pigment to create new color is enhanced by the positive feedback loop, so the warning pigment color can be immediately produced.
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This idea basically mimics the traffic light function: different colors production for every state of the meat. When the meat is still fresh, a specific pigment will be produced. When the meat starts to rot, another pigment will be produced overriding the previous pigment.
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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.

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

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

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



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