http://2012.igem.org/wiki/index.php?title=Special:Contributions/Emeraldo88&feed=atom&limit=50&target=Emeraldo88&year=&month=2012.igem.org - User contributions [en]2024-03-19T11:02:11ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Groningen/StickerTeam:Groningen/Sticker2012-10-27T03:50:25Z<p>Emeraldo88: </p>
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<z1 >The sticker</z1><br />
</div><br />
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<br><br />
<p><br />
Not everyone likes free living, engineered bacteria next to their food. But in order to make our Food Warden<br />
system work properly, our bacteria should be able to react to the volatiles coming off the spoiling meat while it<br />
is located in, for instance, a meat package. We had a lot of discussions about how to achieve a very safe and <br />
easy-in-use solution, and this is what we came up with:<br />
<br><br />
<br> <br />
<i>Bacillus subtilis</i> would be an ideal candidate as a chassis for our genetically engineered construct because it <br />
has the ability to form endospores, a kind of dormant state that they use for survival. They can survive high levels<br />
of heat (>100 °C), drying, radiation, and many damaging chemicals and simply be brought back 'to life' under the influence<br />
of sufficient nutrients and water. This restorative process is germination. For the information, <br />
take a look <a class="inlink" href="http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=69">here</a> and <br />
<a class="inlink" href="http://www.pearsonhighered.com/pearsonhigheredus/educator/product/products_detail.page?isbn=0132324601">here</a>. <br />
Because of this ability to go dormant, our bacterium can be stored and activated whenever it is needed!<br />
<br><br />
<br> <br />
However, this does not solve the problem of the bacteria/spores next to the food. That's why we designed 'the sticker', a <br />
containment device to store and activate the spores at the right time. For clarity: our team keeps calling it 'the sticker' all the time.<br />
The sticker consists of two nested compartments. The inner compartment contains a calibrated amount of nutrients, <br />
while the outer semi-permeable capsule contains the spores of our engineered strain. Breaking the barrier between the two compartments <br />
allows germination and growth of <i>Bacillus subtilis</i> cells. <br />
<br><br><br />
Now the properties of the material we use for our sticker come into play. The polymer we used is TPX®, also<br />
called polymethylpentene. This polymer is available as thin, transparent sheets. The advantage is that it is <br />
relatively cheap, strong, and capable of letting through volatiles. The radius of the pores in TPX® is between <br />
1 nm and 10 nm, which is at least 50x larger than the average badmeat-volatile, but still small enough to keep liquid <br />
and bacteria or spores in (see the figure below). More detailed information about the sticker design and its experiments, are stated below. <br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Siebring J. 2012 (unpublished)<br />
</p><br />
<br><br />
<br><br />
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<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/RR_20120825_tpx.PNG" width="300"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Comparison of the size of the TPX® pores, volatiles and <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<p><br />
<z2>Design requirements</z2><br />
<br><br />
<br><br />
<z3>Material</z3><br />
<br><br />
<br><br />
The material should not break easily and should resist a human grip strength with a minimum of 40 pounds pinch and pressure. <br><br />
The product should be made of a material that is light and easy to handle. <br><br />
The material should be durable and inexpensive.<br> <br />
The volatiles and oxygen should penetrate through the material, while the spores, bacteria and liquid should stay inside.<br><br />
The material should fit in a meat package.<br><br />
The material should not be toxic or become toxic for the bacteria <br><br />
The material should be able to cope with a temperature of at least 125 degrees Celsius.<br />
<br><br />
<br><br />
<z3>Measurements</z3><br />
<br><br />
<br><br />
We want a visible feedback system for the human eye that should easy to understand for the consumer. <br><br />
The visible feedback should not degrade over time.<br />
<br><br />
<br><br />
<z3>Appearance</z3><br />
<br><br />
<br><br />
Product should be have attractive color(s) and recognizable shape.<br />
<br> <br />
<br><br />
<z3>Safety</z3><br />
<br><br />
<br><br />
The bacteria should not escape from the sticker nor harm the environment or the costumer. <br />
Therefore the product should provide adequate support and be reliable.<br />
<br><br />
<br><br />
<z3>Customer comfort</z3><br />
<br><br />
<br><br />
Easy to use the visible feedback device. <br />
<br><br />
The product may provide different degrees of strength depending on the development of the consumer strength. <br />
<br><br />
<br><br />
<z2>Development design</z2><br />
<br><br />
<br><br />
For the development of the sticker, the PDCA (Plan Do Check Act) strategy was used to develop an ultimate <br />
device that keeps track of the needs of the customer and the environment in the project design.<br />
<br><br />
<br> <br />
The following questions where raised: <br />
<br><br />
<br><br />
Who is your external customer?<br />
<br><br />
The people that buy meat on regular basis. <br />
<br><br />
<br> <br />
What value do you want to deliver to that customer?<br />
<br><br />
A replacement for the "use by date and sell by date" system. We want to develop a new product that <br />
indicates when meat starts to spoil before the costumer can notice this by eye, by smell or by taste.<br />
<br> <br />
<br><br />
Who, in you iGEM group, delivers that value?<br />
<br><br />
The design engineers and the biologists of iGEM Groningen 2012.<br />
<br><br />
<br><br />
How do they deliver that value?<br><br />
By doing research and creating a new indicator to predict when meat starts to spoil.<br />
<br><br />
<br><br />
<z3>Plan</z3><br />
<br><br />
<br><br />
Our first plan is to identify exactly what we have to do to make a sticker. <br />
The sticker should be the same as the use of a glow-in-the-dark stick, that you first have to<br />
break before it gives off a bright chemical light. Everyone should understand how <br />
it works. The indication color might be made with the same colors as a traffic light, to <br />
indicate when the meat is fresh, a bit spoiled or really spoiled. During our second case, <br />
we want to produce an easily activated sticker. In the third part of the plan, the outer sticker should be strong enough, <br />
let volatiles go through the outer layer of the sticker, and not any bacteria or liquid should go<br />
out of the outer layer of the sticker.<br />
<br><br />
<br><br />
</p><br />
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<br><br />
<br><br />
<br> <br />
<p><br />
<z3>Do</z3><br />
<br><br />
<br><br />
The following solution was chosen:<br />
Build a transparent plastic bag with a smaller compartment inside, where the medium inside is visible for the user when the color changes.<br />
Make the inner compartment of a weaker material where the sides break when a pinch force is applied.<br />
<br><br />
<br><br />
The following pilots where made:<br><br />
<br><br />
1. Make sticker user-friendly concerning the breakage of the inner compartment.<br><br />
2. Test if the bacteria grow in the sticker.<Br><br />
3. Storage and activation in the sticker.<br><br />
4. Test if the medium with the Food Warden bacteria will produce pigment in the sticker. <br><br />
5. We made a set-up with pumps to check the influence of oxygen poor and oxygen rich environments on the sticker. <br><br />
6. Oxygen concentration tested in jars with rotten meat at different temperatures.<br><br />
<br />
<br><br />
<br><br />
<z3>Check</z3><br />
<br><br />
<br><br />
Depending on the success of the pilots, the number of areas for improvements and the scope of the whole initiative,<br />
we decided to repeat the "Do" and "Check" phases, incorporating our additional improvements.<br />
<br><br />
<br><br />
Once you are finally satisfied that the costs would outweigh the benefits of repeating the Do-Check sub-cycle,<br />
you can move on to the final phase.<br />
<br><br />
<br><br />
<z3>Act</z3><br />
<br><br />
<br><br />
After implementing our solution, we generated part of a continuous improvement initiative, we made a loop back to the Plan Phase<br />
and seek out further areas for improvement.<br />
<br> <br />
<br><br />
<z2>Material</z2><br />
<br><br />
<br><br />
Two different materials were carefully chosen for our final product. At first, the outer <br />
compartment should be resistant enough to support the bacteria inside the sticker and give <br />
resistance to the pressure that is applied by the customer. Its physical properties and characteristics<br />
should not be affected when too much pressure or deformation is applied on the sticker. It should be<br />
light and small, to ensure the capsule is easily placed in meat package for instance.<br />
Second, the case should be made from a light material and the surface should be flat to provide grip <br />
and avoid sharp edges or any other risks for the user.<br />
<br><br />
<br><br />
Taking into account all these characteristics, we considered three materials for the sticker: <br />
polyetheen (sandwich bag), Polyvinyl chloride (cling film) and Polymethylpentene (PMP) also commonly<br />
known as TPX®. We chose TPX® as the most suitable material for the outer layer of the sticker, because <br />
this material fulfills all the requirements mentioned above. <br />
<br> <br />
<br><br />
In addition, we searched for another material needed for the inner compartment of the sticker, <br />
that separates the growth medium from the spores untill the consumer wants to use it. At first, <br />
polyvinyl chloride turned out to be too weak for the outer layer of the sticker, because it is easily broken.<br />
However, these properties are very suitable for the breakable inner compartment of the sticker.<br />
Very little pressure is needed to break the inner compartment, so it will be easy to start <br />
the Food Warden system when the consumer needs it.<br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Krentsel B.A., Kissin Y.V., Kleiner V.I., Stotskaya S.S. Polymers and Copolymers of Higher a-Olefins, Hanser Publishers: New York, 1997.<br><br />
2. H. C. Raine, J. Appl. Polym. Sci. 11, 39 (1969)<br><br />
3. Mitsui Chemicals Co., Properties of Standard TPX Grades, 2004.<br><br />
4. FDA CFR Title 21 Sec. 177.1520 Olefin polymers (C) 3.3b for TPX(4-methylpentene-1-based olefin copolymer).<br><br />
5. Mathiowetz V, Kashman N, Volland G, Weber K, Dowe M, Rogers S (February 1985). "Grip and pinch strength: normative data for adults". Arch Phys Med Rehabil 66 (2): 69–74. PMID 3970660.<br><br />
</p><br />
<p><br />
<br><br />
<br><br />
<z2>Tools and supplies</z2><br />
<ol class="ref"><br />
<li>Sealboy type: 236 SBSA-2, Serie: 0070133017 to affix the TPX material and cling film in the setting 7.</li><br />
<li>Sanyo labo autoclave MLS-3020U (RUG Serial: 4495) to sterilize the TPX material.</li><br />
<li>Gas profile 1 SCS, Part: 6.103.000, series: 0213372 to work sterile during fill-up the sticker.</li><br />
<li>B-D Plastipak 21G 1<sup>1/2</sup> 40/8 NR2 2ml to fill up the sticker.</li> <br />
<li>Cling film brand: Folia vershoudfolie, 50m width 29cm, 15 micrometer, oxygen permeability, fat- and waterproof. Inner compartment for the the sticker.</li> <br />
<li>TPX X-44B#25 and X-44#50, Company: Mitsui Chemicals Co., Brand: TPX to use as outer layer from the sticker.</li><br />
<li>Luria Broth Brand: Lennox.</li><br />
</ol><br />
<br><br />
</p><br />
<p><br />
<z2>Prototype</z2><br />
<br><br />
<br> <br />
The prototype of the inner compartment is made from cling film with a side affix of 1 mm thickness. <br />
The subsequent created inner compartment was filled with sterilized Luria Broth (LB). In the outer layer of the sticker, <br />
made of TPX®, was filled with the cling film package and the bacterial spores. An important feature that we had to take into <br />
account was the oxygen exchange between the TPX® and the LB growth medium. If this does not occur extensively, <br />
the bacteria will not grow at their optimal rate. Therefore, we did several experiments to calculate the minimum <br />
oxygen exchange. We used closed flasks containing 50ml medium and 84ml air to test the bacterial growth at 37 degrees Celsius. <br />
The 50 ml medium in the flasks had a diameter with an average of 6 cm where 84ml of air average of 21% oxygen at a starting point <br />
is applied on the medium in the flask. That makes 17.64ml of total oxygen that is needed for the growth of 50ml medium. <br />
From the TPX® characteristics and mathematical calculations we made the following graph.<br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_ID_20120924_Foodwarden_sticker_surface_graph.png" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
This graph represents the minimum surface cm<sup>2</sup> is needed per ml. The green line is TPX® 50 um thickness and the red line is<br />
25um thickness. The x-axis is the volume of ml Luria Broth containing the Food Warden bacterium. <br />
The y-axis is the required surface area (cubic cm) per mL at 37 degrees Celsius.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p><br />
<br><br />
<z2>Test</z2><br />
<br><br />
<br><br />
<br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_ID_20120924_Sticker_diffrent_volumes_COrr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
We want to check if each different volume is equally easy to break.<br />
</p><br />
<td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<iframe left="150" width="500" height="400" src="http://www.youtube.com/embed/rlaCpIiV-24"></iframe><br />
<br><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
In the movie we see how easy the sticker is to break. <br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<p><br />
<br><br />
Test if the bacteria grow in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_ID_20120924_Sticker_diffrent_volumes_before_growCorr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
All different volumes just after breakage, the LB broth has no traces of bacterial growth.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Bacteria grow inside the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/8/83/Groningen2012_ID_20120924_Sticker_diffrent_volumes_grow_Corr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
We tested also different kinds of volumes to verify the calculations in the surface area plot.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><p><br />
Storage and activation of Bacillus subtilis spores<br><br />
<br><br />
<br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f4/Groningen2012_ID_20121026_sticker_with_spores.jpg" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="500px"><br />
<br><p><br />
Spores placed in the sticker.<br />
</p> <br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br><p><br />
One of bases of our project is the idea that spores can be stored inside the sticker and activated when needed. However, germination only occurs when conditions are favorable for the bacteria to grow. To test whether this condition can be made inside the sticker a very simple experiment was done: germination of spores inside a sticker. Several stickers were made that contained B. subtilis spores in one compartment and normal luria broth medium in the second compartment. The sticker was stored for 1 day to prove that it is able to store them. On the second day, the compartment with medium was broken, thus mixing the medium with the spores. After a day on room temperature, the growth of B. subtilis inside the stickers was observed. This proves that the spores can be stored inside the sticker and that they can be activated at will.</p><br><br />
<br />
<p><br />
<br><br />
Test if the medium with the Food warden bacteria will produce pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Groningen2012_ID_20120927_Sticker_before_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
No growth in the sticker when it was placed in a bottle with rotten meat first (probably due to oxygen depletion), <br />
therefore we placed the sticker in a bottle with fresh meat<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra - Test if the medium with the Food warden bacteria will produce a pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_ID_20120925_sticker_test.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Bottle of rotten meat connected to the microarray set up to ensure the oxygen is pumped around<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br> <br />
<br />
<p><br />
<br><br />
Test if the medium with the Food Warden bacteria will produce a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/49/Groningen2012_ID_20120927_Sticker_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
First picture of the <i>Bacillus</i> producing the purple AmilCP pigment in the sticker and not in a bottle of the microarray set up.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra - Test if the medium with the Food Warden bacteria will produce a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<p><br />
Sticker oxygen input test setup<br><br />
To test the stickers with different amounts of oxygen supplied, a system was built with two pumps. One pump was used for the regular flow of meat volatiles. The other pump was used for pumping fresh air into the flask with the stickers. These pumps alternate in activity so that every 15 minutes fresh air is supplied for 3 minutes. Two different systems were made, one where the air input is applied directly into a jar with stickers and one where the jar with stickers is placed at a different spot so it obtains some fresh air but far less. The setup is shown below:<br />
</p><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/Groningen2012_ID_stickerpumpsetup.jpg" width="800"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="500px"><br />
<br><br />
<p><br />
Left: pump 1 is turned on. The air flows through the system as is indicated by the arrows. Both flasks with stickers are provided with the same amount of volatiles from the meat. Pump 2 (turned off) is connected to the fresh air. Right: Pump 2 is turned on. Fresh air flows in the flask with stickers as is indicated with the arrows. The other flask only receives a small amount of fresh air when pump 1 is turned back on and the remaining air is pumped through the whole system.<br />
</p><br />
<br><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<p><br />
We also used stickers with different plastic thickness. The tested stickers were 25µm and 50µm. The results show a clear result that the stickers supplied with plenty of fresh air and a thickness of 25µm already produce color in 1 day. The stickers with the thicker plastic produced color one day later. For the low fresh air supply color was produced after 3 days with the 25µm plastic. The 50µm plastic took one day longer to produce color.<br />
</p><br />
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<br><br />
<br><br />
<p><br />
The first series of sticker experiments let us believe that oxygen limitation might play a role in the growth of <i>B.subtilis</i> inside the sticker. We devised an experimental setup that would allow us to monitor the oxygen level inside a closed system with rotten meat. Doing triplo tests, in which a 100 ml bottle filled with about 30 grams of meat is placed at 20 or 37°C. By taking a 1ml air sample from the bottle each hour the oxygen level of this closed system is measured for both conditions over time. Each hour the air sample was injected in a closed chamber with a Clark electrode. This gas-phase oxygen electrode system makes a measurement of the oxygen level. The oxygen sample was analyzed with a certificated ISO 6141 from the Linde group, using 21% and 2% oxygen as references and N2 as a base line reference. The data was taken over 24 hours and plotted in a graph. From this graph it was noticed that the oxygen levels drop from 21% to ≈11% in a 20°C environment and to ≈3% in a 37°C environment. Because our initial sticker design was based on a 21% oxygen environment, a change of the surface size of the sticker is needed to ensure enough oxygen exchange inside the sticker. In the second series of sticker tests, we already made the surface larger to prevent this limitation from giving any problems during the testing. Using this data we can design stickers that can compensate for lower oxygen levels, thus ensuring that there is no limitation on germination, growth and pigment production.<br />
</p><br />
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<table class="centertable"><br />
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<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Groningen2012_ID_20121026_oxygen_meat_test_20C_37C.png" width="600"><br />
</td><br />
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<tr><br />
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<td align="center" width="500px"><br />
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<p><br />
Oxygen percentage graph from meat in 20°C and 37°C.<br />
</p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/StickerTeam:Groningen/Sticker2012-10-27T03:47:32Z<p>Emeraldo88: </p>
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<z1 >The sticker</z1><br />
</div><br />
</div><br />
<br><br />
<p><br />
Not everyone likes free living, engineered bacteria next to their food. But in order to make our Food Warden<br />
system work properly, our bacteria should be able to react to the volatiles coming off the spoiling meat while it<br />
is located in, for instance, a meat package. We had a lot of discussions about how to achieve a very safe and <br />
easy-in-use solution, and this is what we came up with:<br />
<br><br />
<br> <br />
<i>Bacillus subtilis</i> would be an ideal candidate as a chassis for our genetically engineered construct because it <br />
has the ability to form endospores, a kind of dormant state that they use for survival. They can survive high levels<br />
of heat (>100 °C), drying, radiation, and many damaging chemicals and simply be brought back 'to life' under the influence<br />
of sufficient nutrients and water. This restorative process is germination. For the information, <br />
take a look <a class="inlink" href="http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=69">here</a> and <br />
<a class="inlink" href="http://www.pearsonhighered.com/pearsonhigheredus/educator/product/products_detail.page?isbn=0132324601">here</a>. <br />
Because of this ability to go dormant, our bacterium can be stored and activated whenever it is needed!<br />
<br><br />
<br> <br />
However, this does not solve the problem of the bacteria/spores next to the food. That's why we designed 'the sticker', a <br />
containment device to store and activate the spores at the right time. For clarity: our team keeps calling it 'the sticker' all the time.<br />
The sticker consists of two nested compartments. The inner compartment contains a calibrated amount of nutrients, <br />
while the outer semi-permeable capsule contains the spores of our engineered strain. Breaking the barrier between the two compartments <br />
allows germination and growth of <i>Bacillus subtilis</i> cells. <br />
<br><br><br />
Now the properties of the material we use for our sticker come into play. The polymer we used is TPX®, also<br />
called polymethylpentene. This polymer is available as thin, transparent sheets. The advantage is that it is <br />
relatively cheap, strong, and capable of letting through volatiles. The radius of the pores in TPX® is between <br />
1 nm and 10 nm, which is at least 50x larger than the average badmeat-volatile, but still small enough to keep liquid <br />
and bacteria or spores in (see the figure below). More detailed information about the sticker design and its experiments, are stated below. <br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Siebring J. 2012 (unpublished)<br />
</p><br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/RR_20120825_tpx.PNG" width="300"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Comparison of the size of the TPX® pores, volatiles and <i>Bacillus subtilis</i>.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<p><br />
<z2>Design requirements</z2><br />
<br><br />
<br><br />
<z3>Material</z3><br />
<br><br />
<br><br />
The material should not break easily and should resist a human grip strength with a minimum of 40 pounds pinch and pressure. <br><br />
The product should be made of a material that is light and easy to handle. <br><br />
The material should be durable and inexpensive.<br> <br />
The volatiles and oxygen should penetrate through the material, while the spores, bacteria and liquid should stay inside.<br><br />
The material should fit in a meat package.<br><br />
The material should not be toxic or become toxic for the bacteria <br><br />
The material should be able to cope with a temperature of at least 125 degrees Celsius.<br />
<br><br />
<br><br />
<z3>Measurements</z3><br />
<br><br />
<br><br />
We want a visible feedback system for the human eye that should easy to understand for the consumer. <br><br />
The visible feedback should not degrade over time.<br />
<br><br />
<br><br />
<z3>Appearance</z3><br />
<br><br />
<br><br />
Product should be have attractive color(s) and recognizable shape.<br />
<br> <br />
<br><br />
<z3>Safety</z3><br />
<br><br />
<br><br />
The bacteria should not escape from the sticker nor harm the environment or the costumer. <br />
Therefore the product should provide adequate support and be reliable.<br />
<br><br />
<br><br />
<z3>Customer comfort</z3><br />
<br><br />
<br><br />
Easy to use the visible feedback device. <br />
<br><br />
The product may provide different degrees of strength depending on the development of the consumer strength. <br />
<br><br />
<br><br />
<z2>Development design</z2><br />
<br><br />
<br><br />
For the development of the sticker, the PDCA (Plan Do Check Act) strategy was used to develop an ultimate <br />
device that keeps track of the needs of the customer and the environment in the project design.<br />
<br><br />
<br> <br />
The following questions where raised: <br />
<br><br />
<br><br />
Who is your external customer?<br />
<br><br />
The people that buy meat on regular basis. <br />
<br><br />
<br> <br />
What value do you want to deliver to that customer?<br />
<br><br />
A replacement for the "use by date and sell by date" system. We want to develop a new product that <br />
indicates when meat starts to spoil before the costumer can notice this by eye, by smell or by taste.<br />
<br> <br />
<br><br />
Who, in you iGEM group, delivers that value?<br />
<br><br />
The design engineers and the biologists of iGEM Groningen 2012.<br />
<br><br />
<br><br />
How do they deliver that value?<br><br />
By doing research and creating a new indicator to predict when meat starts to spoil.<br />
<br><br />
<br><br />
<z3>Plan</z3><br />
<br><br />
<br><br />
Our first plan is to identify exactly what we have to do to make a sticker. <br />
The sticker should be the same as the use of a glow-in-the-dark stick, that you first have to<br />
break before it gives off a bright chemical light. Everyone should understand how <br />
it works. The indication color might be made with the same colors as a traffic light, to <br />
indicate when the meat is fresh, a bit spoiled or really spoiled. During our second case, <br />
we want to produce an easily activated sticker. In the third part of the plan, the outer sticker should be strong enough, <br />
let volatiles go through the outer layer of the sticker, and not any bacteria or liquid should go<br />
out of the outer layer of the sticker.<br />
<br><br />
<br><br />
</p><br />
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<br><br />
<br> <br />
<p><br />
<z3>Do</z3><br />
<br><br />
<br><br />
The following solution was chosen:<br />
Build a transparent plastic bag with a smaller compartment inside, where the medium inside is visible for the user when the color changes.<br />
Make the inner compartment of a weaker material where the sides break when a pinch force is applied.<br />
<br><br />
<br><br />
The following pilots where made:<br><br />
<br><br />
1. Make sticker user-friendly concerning the breakage of the inner compartment.<br><br />
2. Test if the bacteria grow in the sticker.<Br><br />
3. Storage and activation in the sticker.<br><br />
4. Test if the medium with the Food Warden bacteria will produce pigment in the sticker. <br><br />
5. We made a set-up with pumps to check the influence of oxygen poor and oxygen rich environments on the sticker. <br><br />
6. Oxygen concentration tested in jars with rotten meat at different temperatures.<br><br />
<br />
<br><br />
<br><br />
<z3>Check</z3><br />
<br><br />
<br><br />
Depending on the success of the pilots, the number of areas for improvements and the scope of the whole initiative,<br />
we decided to repeat the "Do" and "Check" phases, incorporating our additional improvements.<br />
<br><br />
<br><br />
Once you are finally satisfied that the costs would outweigh the benefits of repeating the Do-Check sub-cycle,<br />
you can move on to the final phase.<br />
<br><br />
<br><br />
<z3>Act</z3><br />
<br><br />
<br><br />
After implementing our solution, we generated part of a continuous improvement initiative, we made a loop back to the Plan Phase<br />
and seek out further areas for improvement.<br />
<br> <br />
<br><br />
<z2>Material</z2><br />
<br><br />
<br><br />
Two different materials were carefully chosen for our final product. At first, the outer <br />
compartment should be resistant enough to support the bacteria inside the sticker and give <br />
resistance to the pressure that is applied by the customer. Its physical properties and characteristics<br />
should not be affected when too much pressure or deformation is applied on the sticker. It should be<br />
light and small, to ensure the capsule is easily placed in meat package for instance.<br />
Second, the case should be made from a light material and the surface should be flat to provide grip <br />
and avoid sharp edges or any other risks for the user.<br />
<br><br />
<br><br />
Taking into account all these characteristics, we considered three materials for the sticker: <br />
polyetheen (sandwich bag), Polyvinyl chloride (cling film) and Polymethylpentene (PMP) also commonly<br />
known as TPX®. We chose TPX® as the most suitable material for the outer layer of the sticker, because <br />
this material fulfills all the requirements mentioned above. <br />
<br> <br />
<br><br />
In addition, we searched for another material needed for the inner compartment of the sticker, <br />
that separates the growth medium from the spores untill the consumer wants to use it. At first, <br />
polyvinyl chloride turned out to be too weak for the outer layer of the sticker, because it is easily broken.<br />
However, these properties are very suitable for the breakable inner compartment of the sticker.<br />
Very little pressure is needed to break the inner compartment, so it will be easy to start <br />
the Food Warden system when the consumer needs it.<br />
<br><br />
<br><br />
</p><br />
<z4>References</z4><br />
<p class="ref"><br />
1. Krentsel B.A., Kissin Y.V., Kleiner V.I., Stotskaya S.S. Polymers and Copolymers of Higher a-Olefins, Hanser Publishers: New York, 1997.<br><br />
2. H. C. Raine, J. Appl. Polym. Sci. 11, 39 (1969)<br><br />
3. Mitsui Chemicals Co., Properties of Standard TPX Grades, 2004.<br><br />
4. FDA CFR Title 21 Sec. 177.1520 Olefin polymers (C) 3.3b for TPX(4-methylpentene-1-based olefin copolymer).<br><br />
5. Mathiowetz V, Kashman N, Volland G, Weber K, Dowe M, Rogers S (February 1985). "Grip and pinch strength: normative data for adults". Arch Phys Med Rehabil 66 (2): 69–74. PMID 3970660.<br><br />
</p><br />
<p><br />
<br><br />
<br><br />
<z2>Tools and supplies</z2><br />
<ol class="ref"><br />
<li>Sealboy type: 236 SBSA-2, Serie: 0070133017 to affix the TPX material and cling film in the setting 7.</li><br />
<li>Sanyo labo autoclave MLS-3020U (RUG Serial: 4495) to sterilize the TPX material.</li><br />
<li>Gas profile 1 SCS, Part: 6.103.000, series: 0213372 to work sterile during fill-up the sticker.</li><br />
<li>B-D Plastipak 21G 1<sup>1/2</sup> 40/8 NR2 2ml to fill up the sticker.</li> <br />
<li>Cling film brand: Folia vershoudfolie, 50m width 29cm, 15 micrometer, oxygen permeability, fat- and waterproof. Inner compartment for the the sticker.</li> <br />
<li>TPX X-44B#25 and X-44#50, Company: Mitsui Chemicals Co., Brand: TPX to use as outer layer from the sticker.</li><br />
<li>Luria Broth Brand: Lennox.</li><br />
</ol><br />
<br><br />
</p><br />
<p><br />
<z2>Prototype</z2><br />
<br><br />
<br> <br />
The prototype of the inner compartment is made from cling film with a side affix of 1 mm thickness. <br />
The subsequent created inner compartment was filled with sterilized Luria Broth (LB). In the outer layer of the sticker, <br />
made of TPX®, was filled with the cling film package and the bacterial spores. An important feature that we had to take into <br />
account was the oxygen exchange between the TPX® and the LB growth medium. If this does not occur extensively, <br />
the bacteria will not grow at their optimal rate. Therefore, we did several experiments to calculate the minimum <br />
oxygen exchange. We used closed flasks containing 50ml medium and 84ml air to test the bacterial growth at 37 degrees Celsius. <br />
The 50 ml medium in the flasks had a diameter with an average of 6 cm where 84ml of air average of 21% oxygen at a starting point <br />
is applied on the medium in the flask. That makes 17.64ml of total oxygen that is needed for the growth of 50ml medium. <br />
From the TPX® characteristics and mathematical calculations we made the following graph.<br />
<br><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_ID_20120924_Foodwarden_sticker_surface_graph.png" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><br />
This graph represents the minimum surface cm<sup>2</sup> is needed per ml. The green line is TPX® 50 um thickness and the red line is<br />
25um thickness. The x-axis is the volume of ml Luria Broth containing the Food Warden bacterium. <br />
The y-axis is the required surface area (cubic cm) per mL at 37 degrees Celsius.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p><br />
<br><br />
<z2>Test</z2><br />
<br><br />
<br><br />
<br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/d/de/Groningen2012_ID_20120924_Sticker_diffrent_volumes_COrr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
We want to check if each different volume is equally easy to break.<br />
</p><br />
<td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
<br><br />
Make sticker user-friendly concerning the breakage of the inner compartment.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<iframe left="150" width="500" height="400" src="http://www.youtube.com/embed/rlaCpIiV-24"></iframe><br />
<br><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
In the movie we see how easy the sticker is to break. <br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<p><br />
<br><br />
Test if the bacteria grow in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6c/Groningen2012_ID_20120924_Sticker_diffrent_volumes_before_growCorr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
All different volumes just after breakage, the LB broth has no traces of bacterial growth.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Bacteria grow inside the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/8/83/Groningen2012_ID_20120924_Sticker_diffrent_volumes_grow_Corr.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
We tested also different kinds of volumes to verify the calculations in the surface area plot.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><p><br />
Storage and activation of Bacillus subtilis spores<br><br />
<br><br />
<br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f4/Groningen2012_ID_20121026_sticker_with_spores.jpg" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="500px"><br />
<br><p><br />
Spores placed in the sticker.<br />
</p> <br />
<br />
</td><br />
</tr><br />
</table><br />
<br />
<br><p><br />
One of bases of our project is the idea that spores can be stored inside the sticker and activated when needed. However, germination only occurs when conditions are favorable for the bacteria to grow. To test whether this condition can be made inside the sticker a very simple experiment was done: germination of spores inside a sticker. Several stickers were made that contained B. subtilis spores in one compartment and normal luria broth medium in the second compartment. The sticker was stored for 1 day to prove that it is able to store them. On the second day, the compartment with medium was broken, thus mixing the medium with the spores. After a day on room temperature, the growth of B. subtilis inside the stickers was observed. This proves that the spores can be stored inside the sticker and that they can be activated at will.</p><br><br />
<br />
<p><br />
<br><br />
Test if the medium with the Food warden bacteria will produce pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Groningen2012_ID_20120927_Sticker_before_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
No growth in the sticker when it was placed in a bottle with rotten meat first (probably due to oxygen depletion), <br />
therefore we placed the sticker in a bottle with fresh meat<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra - Test if the medium with the Food warden bacteria will produce a pigment in the sticker.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c1/Groningen2012_ID_20120925_sticker_test.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Bottle of rotten meat connected to the microarray set up to ensure the oxygen is pumped around<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br> <br />
<br />
<p><br />
<br><br />
Test if the medium with the Food Warden bacteria will produce a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/4/49/Groningen2012_ID_20120927_Sticker_worked.png" width="500"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="800px"><br />
<p class="captionnomargin"><br />
First picture of the <i>Bacillus</i> producing the purple AmilCP pigment in the sticker and not in a bottle of the microarray set up.<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<p><br />
<br><br />
Extra - Test if the medium with the Food Warden bacteria will produce a pigment in the sticker.<br />
<br><br />
<br><br />
</p> <br />
<p><br />
Sticker oxygen input test setup<br />
To test the stickers with different amounts of oxygen supplied, a system was built with two pumps. One pump was used for the regular flow of meat volatiles. The other pump was used for pumping fresh air into the flask with the stickers. These pumps alternate in activity so that every 15 minutes fresh air is supplied for 3 minutes. Two different systems were made, one where the air input is applied directly into a jar with stickers and one where the jar with stickers is placed at a different spot so it obtains some fresh air but far less. The setup is shown below:<br />
</p><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/e/e8/Groningen2012_ID_stickerpumpsetup.jpg" width="800"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="500px"><br />
<br><br />
<p><br />
Left: pump 1 is turned on. The air flows through the system as is indicated by the arrows. Both flasks with stickers are provided with the same amount of volatiles from the meat. Pump 2 (turned off) is connected to the fresh air. Right: Pump 2 is turned on. Fresh air flows in the flask with stickers as is indicated with the arrows. The other flask only receives a small amount of fresh air when pump 1 is turned back on and the remaining air is pumped through the whole system.<br />
</p><br />
<br><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<p><br />
We also used stickers with different plastic thickness. The tested stickers were 25µm and 50µm. The results show a clear result that the stickers supplied with plenty of fresh air and a thickness of 25µm already produce color in 1 day. The stickers with the thicker plastic produced color one day later. For the low fresh air supply color was produced after 3 days with the 25µm plastic. The 50µm plastic took one day longer to produce color.<br />
</p><br />
<br><br />
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<br />
<br><br />
<br><br />
<br />
<br><br />
<br><br />
<br> <br><br />
<br><br />
<br> <br><br />
<br><br />
<br><br />
<p><br />
The first series of sticker experiments let us believe that oxygen limitation might play a role in the growth of <i>B.subtilis</i> inside the sticker. We devised an experimental setup that would allow us to monitor the oxygen level inside a closed system with rotten meat. Doing triplo tests, in which a 100 ml bottle filled with about 30 grams of meat is placed at 20 or 37°C. By taking a 1ml air sample from the bottle each hour the oxygen level of this closed system is measured for both conditions over time. Each hour the air sample was injected in a closed chamber with a Clark electrode. This gas-phase oxygen electrode system makes a measurement of the oxygen level. The oxygen sample was analyzed with a certificated ISO 6141 from the Linde group, using 21% and 2% oxygen as references and N2 as a base line reference. The data was taken over 24 hours and plotted in a graph. From this graph it was noticed that the oxygen levels drop from 21% to ≈11% in a 20°C environment and to ≈3% in a 37°C environment. Because our initial sticker design was based on a 21% oxygen environment, a change of the surface size of the sticker is needed to ensure enough oxygen exchange inside the sticker. In the second series of sticker tests, we already made the surface larger to prevent this limitation from giving any problems during the testing. Using this data we can design stickers that can compensate for lower oxygen levels, thus ensuring that there is no limitation on germination, growth and pigment production.<br />
</p><br />
<br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/e/ea/Groningen2012_ID_20121026_oxygen_meat_test_20C_37C.png" width="600"><br />
</td><br />
</tr><br />
<tr><br />
<tr><br />
<td align="center" width="500px"><br />
<br><br />
<p><br />
Oxygen percentage graph from meat in 20°C and 37°C.<br />
</p><br />
<br><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br><br />
<br><br />
<br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/volatiles"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T23:27:03Z<p>Emeraldo88: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
<br />
<br />
<br />
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<head><br />
<style><br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. <br />
When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the <br />
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced<br />
when the promoter is activated.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration<br />
and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <br />
<i>E. coli</i>.<br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we were back in the lab to build our planned constructs in the<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. <br />
We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. <br />
We engineered the construct inside psac-cm backbone (BBa_K818000)<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
AmilGFP under regulation of P<i>wap</i>A<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
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 <br />
(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 <br />
fresh meat but absent in the presence of rotten meat. <br />
</p><br />
<div class="cte2"><br />
<div class="ctd2"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
<z2>SboA-AmilGFP</z2><br />
<br><br />
<br><br />
<z3>Expression in <i>E. coli</i></z3><br />
<br><br />
<br><br />
<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, <br />
the yellow color is less visible compared to the cell pellet in liquid culture.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"><br />
<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.<br />
</p> <br />
<p><br />
<br><br />
<z3>Expression in <i>B. subtilis</i></z3><br />
<br><br />
<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). <br />
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to<br />
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and<br />
<i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. <br />
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:<br />
<i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), <br />
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 <br />
yellow color at all.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"><br />
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
(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><br />
(right) Pelleted cells after 16 hour growth with/without spoiled meat. <br />
</p><br />
<p><br />
<br><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 <br />
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<br />
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: <br />
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster <br />
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"><br />
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(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><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. <br />
</p> <br />
<br> <br />
<p><br />
<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> <br />
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> <br />
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 <br />
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<br />
was highly upregulated, thus more amilGFP was expressed.<br />
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 <br />
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 /><br />
<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 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
<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.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
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).<br />
</p><br />
<p><br />
<br><br />
<z2>SboA-AmilCP</z2><br />
<br><br />
<br><br />
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 <br />
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
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.<br />
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.<br />
<br><br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br> <br />
<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 />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a3/EJ_Groningen2012_RR-pretty_graphs.png" width=700 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/a3/EJ_Groningen2012_RR-pretty_graphs.png" class="preview" width=1200 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<td align="center"><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><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. <br />
</p><br />
</td><br />
</tr><br />
<br><br><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/EJ_Groningen2012_RR-intensity_over_time.PNG" width=350 /><br />
<img src="https://static.igem.org/mediawiki/2012/f/f8/EJ_Groningen2012_RR-intensity_over_time.PNG" class="preview" width=550 /><br />
</a><br />
</li><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/EJ_Groningen2012_RR-OD_measurement.PNG" width=321 /><br />
<img src="https://static.igem.org/mediawiki/2012/0/0c/EJ_Groningen2012_RR-OD_measurement.PNG" class="preview" width=521 /><br />
</a><br />
</li> <br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<td align="center"><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><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.<br />
</p><br />
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{{Template:SponsorsGroningen2012}}<br />
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<a href="https://2012.igem.org/Team:Groningen/Kill_Switch"><br />
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<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
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</html></div>Emeraldo88http://2012.igem.org/File:EJ_Groningen2012_RR-OD_measurement.PNGFile:EJ Groningen2012 RR-OD measurement.PNG2012-10-26T23:20:21Z<p>Emeraldo88: </p>
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<div></div>Emeraldo88http://2012.igem.org/File:EJ_Groningen2012_RR-intensity_over_time.PNGFile:EJ Groningen2012 RR-intensity over time.PNG2012-10-26T23:17:47Z<p>Emeraldo88: </p>
<hr />
<div></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T23:15:08Z<p>Emeraldo88: </p>
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<div>{{HeaderGroningen2012}}<br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. <br />
When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the <br />
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced<br />
when the promoter is activated.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration<br />
and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <br />
<i>E. coli</i>.<br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we were back in the lab to build our planned constructs in the<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. <br />
We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. <br />
We engineered the construct inside psac-cm backbone (BBa_K818000)<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
AmilGFP under regulation of P<i>wap</i>A<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
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 <br />
(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 <br />
fresh meat but absent in the presence of rotten meat. <br />
</p><br />
<div class="cte2"><br />
<div class="ctd2"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
<z2>SboA-AmilGFP</z2><br />
<br><br />
<br><br />
<z3>Expression in <i>E. coli</i></z3><br />
<br><br />
<br><br />
<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, <br />
the yellow color is less visible compared to the cell pellet in liquid culture.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"><br />
<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.<br />
</p> <br />
<p><br />
<br><br />
<z3>Expression in <i>B. subtilis</i></z3><br />
<br><br />
<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). <br />
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to<br />
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and<br />
<i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. <br />
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:<br />
<i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), <br />
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 <br />
yellow color at all.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"><br />
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
(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><br />
(right) Pelleted cells after 16 hour growth with/without spoiled meat. <br />
</p><br />
<p><br />
<br><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 <br />
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<br />
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: <br />
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster <br />
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"><br />
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(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><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. <br />
</p> <br />
<br> <br />
<p><br />
<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> <br />
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> <br />
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 <br />
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<br />
was highly upregulated, thus more amilGFP was expressed.<br />
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 <br />
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 /><br />
<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 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
<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.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
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).<br />
</p><br />
<p><br />
<br><br />
<z2>SboA-AmilCP</z2><br />
<br><br />
<br><br />
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 <br />
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
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.<br />
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.<br />
<br><br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br> <br />
<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 />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/a3/EJ_Groningen2012_RR-pretty_graphs.png" width=700 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/a3/EJ_Groningen2012_RR-pretty_graphs.png" class="preview" width=1200 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<td align="center"><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><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. <br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<p><br />
<br />
</p><br />
<br><br />
<br><br />
<br><br />
<br><br />
</body><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<a href="https://2012.igem.org/Team:Groningen/Kill_Switch"><br />
<div style="position:absolute; right: 0px; bottom: 760px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T23:01:22Z<p>Emeraldo88: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<body><br />
<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. <br />
When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the <br />
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced<br />
when the promoter is activated.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration<br />
and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <br />
<i>E. coli</i>.<br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we were back in the lab to build our planned constructs in the<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. <br />
We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. <br />
We engineered the construct inside psac-cm backbone (BBa_K818000)<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
AmilGFP under regulation of P<i>wap</i>A<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
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 <br />
(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 <br />
fresh meat but absent in the presence of rotten meat. <br />
</p><br />
<div class="cte2"><br />
<div class="ctd2"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
<z2>SboA-AmilGFP</z2><br />
<br><br />
<br><br />
<z3>Expression in <i>E. coli</i></z3><br />
<br><br />
<br><br />
<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, <br />
the yellow color is less visible compared to the cell pellet in liquid culture.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"><br />
<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.<br />
</p> <br />
<p><br />
<br><br />
<z3>Expression in <i>B. subtilis</i></z3><br />
<br><br />
<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). <br />
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to<br />
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and<br />
<i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. <br />
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:<br />
<i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), <br />
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 <br />
yellow color at all.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"><br />
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
(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><br />
(right) Pelleted cells after 16 hour growth with/without spoiled meat. <br />
</p><br />
<p><br />
<br><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 <br />
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<br />
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: <br />
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster <br />
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"><br />
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(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><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. <br />
</p> <br />
<br> <br />
<p><br />
<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> <br />
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> <br />
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 <br />
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<br />
was highly upregulated, thus more amilGFP was expressed.<br />
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 <br />
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 /><br />
<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 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
<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.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
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).<br />
</p><br />
<p><br />
<br><br />
<z2>SboA-AmilCP</z2><br />
<br><br />
<br><br />
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 <br />
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
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.<br />
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.<br />
<br><br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br> <br />
<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 />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a3/EJ_Groningen2012_RR-pretty_graphs.png/800px-EJ_Groningen2012_RR-pretty_graphs.png" width="350"><br />
</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><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. <br />
</p><br />
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</td><br />
</tr><br />
<tr><br />
<td align="center"><br />
<p class="captionnomargin"><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. <br />
</p><br />
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</table><br />
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{{Template:SponsorsGroningen2012}}<br />
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<div style="position:absolute; right: 0px; bottom: 760px;"><br />
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</div><br />
</a><br />
<div style="position:absolute; right: 10px; bottom: 700px;"><br />
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</html></div>Emeraldo88http://2012.igem.org/File:EJ_Groningen2012_RR-pretty_graphs.pngFile:EJ Groningen2012 RR-pretty graphs.png2012-10-26T22:57:14Z<p>Emeraldo88: </p>
<hr />
<div></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T22:55:23Z<p>Emeraldo88: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. <br />
When <i>Bacillus subtilis</i> senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the <br />
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced<br />
when the promoter is activated.<br />
<br><br />
</p><br />
<table class="centertable"><br />
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<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/5/55/Groningen2012_EJ_20120912_psaccmt-RFP-contruct-edited.png" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
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<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
Hover your mouse over the image to see a bigger version!<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
We use our <i>Bacillus subtilis</i> backbone (BBa_K818000) that has <i>sacA</i> and a chloramphenicol resistance gene for chromosomal integration<br />
and antibiotic screening of transformants respectively. This backbone also has <i>E. coli</i> origin of replication, so it can be amplified inside <br />
<i>E. coli</i>.<br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br><br />
After the European regional jamboree, we were back in the lab to build our planned constructs in the<br />
<a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. <br />
We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. <br />
We engineered the construct inside psac-cm backbone (BBa_K818000)<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
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<td align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"><br />
</td><br />
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<tr><br />
<td align="center"><br />
<p class="captionnomargin"><br />
AmilGFP under regulation of PwapA<br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<p><br />
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 <br />
(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 <br />
fresh meat but absent in the presence of rotten meat. <br />
</p><br />
<div class="cte2"><br />
<div class="ctd2"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<p><br />
<br><br />
<z2>SboA-AmilGFP</z2><br />
<br><br />
<br><br />
<z3>Expression in <i>E. coli</i></z3><br />
<br><br />
<br><br />
<i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, <br />
the yellow color is less visible compared to the cell pellet in liquid culture.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"><br />
<img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
(right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP.<br />
</p> <br />
<p><br />
<br><br />
<z3>Expression in <i>B. subtilis</i></z3><br />
<br><br />
<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). <br />
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to<br />
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and<br />
<i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. <br />
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:<br />
<i>B. subtilis</i> sboA-AmilGFP strain that was subjected to spoiled meat had turned bright greenish yellow (even visible in liquid LB culture), <br />
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 <br />
yellow color at all.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"><br />
<img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
(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><br />
(right) Pelleted cells after 16 hour growth with/without spoiled meat. <br />
</p><br />
<p><br />
<br><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 <br />
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<br />
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: <br />
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster <br />
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.<br />
<br><br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"><br />
<img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
(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><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. <br />
</p> <br />
<br> <br />
<p><br />
<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> <br />
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> <br />
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 <br />
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<br />
was highly upregulated, thus more amilGFP was expressed.<br />
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 <br />
the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment.<br />
<br><br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/thumb/a/a6/Groningen2012_Overview_microscopy.png/641px-Groningen2012_Overview_microscopy.png" width=400 height=257 /><br />
<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 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table><br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
<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.<br />
</p><br />
<table class="centertable"><br />
<tr><br />
<td align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><br />
<img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /><br />
</a><br />
</li><br />
</ul><br />
</td><br />
</tr><br />
</table> <br />
<p class="caption"><br />
Hover your mouse over the image to see a bigger version!<br><br />
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).<br />
</p><br />
<p><br />
<br><br />
<z2>SboA-AmilCP</z2><br />
<br><br />
<br><br />
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 <br />
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
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.<br />
See the <a class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information.<br />
<br><br />
<br><br />
<br><br />
<z2>Update! (26th October 2012)</z2><br />
<br><br />
<br> <br />
<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 />
<br />
<br />
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</a><br />
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</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_26October2012Team:Groningen/Notebook/Wetwork 26October20122012-10-26T20:53:22Z<p>Emeraldo88: </p>
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Oxygen tests<br><br />
<br />
And Wikifreeze...<br />
<br><br />
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<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A><br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_22October2012Team:Groningen/Notebook/Wetwork 22October20122012-10-26T19:59:41Z<p>Emeraldo88: </p>
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Done growthcurves for the modeling the whole day. Duplo experiments with <i>Bacillus subtilis</i> transformed with BBa_K818600 at room temperatue, 30 degrees Celsius and 37 degrees Celsius<br><br><br />
Plasmid isolation from yesterday's O/N culture, cut with EcoRI and PstI for length check (psaccm-wapA-eforRed, psaccm-wapA-amilGFP, psaccm-sboA-eforRed). The correct sizes were then transformed into <i>B. subtilis</i>. Transformants were then plated on chloramphenicol LB plates.<br />
<br><br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_21October2012Team:Groningen/Notebook/Wetwork 21October20122012-10-26T19:56:18Z<p>Emeraldo88: Created page with "{{HeaderGroningen2012}} <html> <head> <style type="text/css"> p.margin { font-size:12pt; line-height:14pt; color:white; margin-top:0px; margin-bottom:20px; margin-left:150px..."</p>
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O/N culture of the transformants and O/N of <i>Bacillus subtilis</i> 168 wild type for transformation tomorrow.<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_20October2012Team:Groningen/Notebook/Wetwork 20October20122012-10-26T19:52:44Z<p>Emeraldo88: Created page with "{{HeaderGroningen2012}} <html> <head> <style type="text/css"> p.margin { font-size:12pt; line-height:14pt; color:white; margin-top:0px; margin-bottom:20px; margin-left:150px..."</p>
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Plasmid isolation of psaccm-eforRed, cut with EcoRI and PstI for insert check, cut with EcoRI and XbaI for ligation reaction later with cut PwapA and PsboA with EcoRI and SpeI. Cut amilGFP with EcoRI and XbaI for ligation with PwapA. Ligation reaction of PwapA with eforRed and amilGFP, PsboA with eforRed. Transformation of <i>E. coli</i> with ligated product.<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_19October2012Team:Groningen/Notebook/Wetwork 19October20122012-10-26T19:45:02Z<p>Emeraldo88: Created page with "{{HeaderGroningen2012}} <html> <head> <style type="text/css"> p.margin { font-size:12pt; line-height:14pt; color:white; margin-top:0px; margin-bottom:20px; margin-left:150px..."</p>
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O/N culture of the transformants from yesterday's <i>E. coli</i> transformation. wapA PCR for tomorrow's ligation with cut eforRed and cut amilGFP.<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_18October2012Team:Groningen/Notebook/Wetwork 18October20122012-10-26T19:42:28Z<p>Emeraldo88: </p>
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Plasmid isolation from the O/N culture of the transformants. All of the plasmids were cut with EcoRI and PstI to check for the inserts' length. Only the eforRed plasmids showed the correct insert's bands. The eforRed band was then purified from the gel and ligated into Psaccm. The ligated product was then used to transform <i>E. coli</i> DH5alpha, plated into ampicillin LB agar for O/N 37 degree incubation.<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_18October2012Team:Groningen/Notebook/Wetwork 18October20122012-10-26T19:36:24Z<p>Emeraldo88: Created page with "{{HeaderGroningen2012}} <html> <head> <style type="text/css"> p.margin { font-size:12pt; line-height:14pt; color:white; margin-top:0px; margin-bottom:20px; margin-left:150px..."</p>
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Plasmid isolation from the O/N culture of the transformants. All of the plasmids were cut with EcoRI and PstI to check for the inserts' length. Only the eforRed plasmids showed the correct insert's bands. The eforRed band was then purified from the gel and ligated into Psaccm<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_17October2012Team:Groningen/Notebook/Wetwork 17October20122012-10-26T19:17:07Z<p>Emeraldo88: </p>
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Tests were done with the construct psaccm-SboA-AmilGP with rotten and fresh meat in duplo. Samples were obtained every hour and plated in three different dilutions. Cells were spun down and resuspended in sz. The resuspended cells were analyzed with flowcytometrie and with a spectrophotometer for measuring OD (600nm) and color (359nm).<br><br><br />
Overnight culture of the transformants from yesterday's <i>E. coli</i> transformation.<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_16October2012Team:Groningen/Notebook/Wetwork 16October20122012-10-26T19:15:40Z<p>Emeraldo88: </p>
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<b>Emeraldo</b><br><br />
Transformation of <i>E. coli</i> DH5alpha with plasmid PSB1C3 with eforRed (red pigment) and aeBlue (blue pigment) from Uppsalla-Sweden. <br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_16October2012Team:Groningen/Notebook/Wetwork 16October20122012-10-26T19:15:21Z<p>Emeraldo88: Created page with "{{HeaderGroningen2012}} <html> <head> <style type="text/css"> p.margin { font-size:12pt; line-height:14pt; color:white; margin-top:0px; margin-bottom:20px; margin-left:150px..."</p>
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<b>Emeraldo<b><br />
Transformation of <i>E. coli</i> DH5alpha with plasmid PSB1C3 with eforRed (red pigment) and aeBlue (blue pigment) from Uppsalla-Sweden. <br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/Notebook/Wetwork_10October2012Team:Groningen/Notebook/Wetwork 10October20122012-10-26T19:08:58Z<p>Emeraldo88: Created page with "{{HeaderGroningen2012}} <html> <head> <style type="text/css"> p.margin { font-size:12pt; line-height:14pt; color:white; margin-top:0px; margin-bottom:20px; margin-left:150px..."</p>
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Designing primer for <i>wap</i>A promoter and <i>mnt</i>R promoter. The designed primers were then ordered.<br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T18:52:42Z<p>Emeraldo88: </p>
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<z1>Future Plans</z1><br />
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We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
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<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
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The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
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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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
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When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
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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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
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<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
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<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
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<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a new construct: AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). The construct is a pilot construct for the following diagram: pigment 1 is regulated by the rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). <br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
When meat is still fresh, the P<i>wap</i>A will regulate a production of pigment 1 while pigment 2 is absent, regulated by P<i>sbo</i>A in fresh meat conditions. When the meat is rotten, P<i>wap</i>A will be down-regulated, thus decreasing the production of pigment 1 and P<i>sbo</i>A will be up-regulated, producing pigment 2. For example: If pigment 1 is a yellow pigment (AmilGFP) and pigment 2 is a blue pigment (amilCP), a strong yellow color will be produced when the meat is still fresh and when the meat is rotten, more blue pigment will be produced. So when the meat is rotten, a green color pigment is obtained. <br />
</p><br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T18:49:00Z<p>Emeraldo88: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Future Plans</z1><br />
</div><br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a new construct: AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). The construct is a pilot construct for the following diagram: pigment 1 is regulated by the rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). <br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
When meat is still fresh, the P<i>wap</i>A will regulate a production of pigment 1 while pigment 2 is absent, regulated by P<i>sbo</i>A in fresh meat conditions. When the meat is rotten, P<i>wap</i>A will be down-regulated, thus decreasing the production of pigment 1 and P<i>sbo</i>A will be up-regulated, producing pigment 2. For example: If pigment 1 is a yellow pigment (AmilGFP) and pigment 2 is a red pigment (lycopene), a strong yellow color will be produced when the meat is still fresh and when the meat is rotten, more red will be obtained. So when the meat is rotten, an orange color is obtained. <br />
</p><br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T18:32:52Z<p>Emeraldo88: </p>
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<br><br />
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<z1>Future Plans</z1><br />
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<br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to construct the amilGFP (yellow pigment) under regulation of P<i>wap</i>A (down-regulated promoter by the presence of rotten meat). This construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
Pigment 1 is regulated by down-regulated promoter in the presence of rotten meat, in this case wapA, and pigment 2 is regulated by the up-regulated promoter (sboA). When the meat is still fresh, the P<i>wap</i>A will regulate a higher production of pigment 1 compared to the pigment 2 production regulated by P<i>sbo</i>A. The pigment 1 will be produced more than the pigment 2 in the fresh meat condition. When the meat is rotten, P<i>wap</i>A will be down-regulated thus decreasing the production of pigment 1 and the P<i>sbo</i>A will be up-regulated, increasing the production of pigment 2. For example: If the pigment 1 is a yellow pigment (amilGFP) and the pigment 2 is a blue pigment (AmilCP), a strong yellow color will be produced when the meat is still fresh. When the meat is rotten, the pigment will be more blue because the production of the yellow pigment stopped and the blue pigment will be up-regulated, showing greenish-blue color. <br />
</p><br />
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<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T18:29:15Z<p>Emeraldo88: </p>
<hr />
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<z1>Future Plans</z1><br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to construct the AmilGFP (yellow pigment) under regulation of P<i>wap</i>A (down-regulated promoter by the presence of rotten meat). This construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
Pigment 1 is regulated by down-regulated promoter in the presence of rotten meat, in this case wapA, and pigment 2 is regulated by the up-regulated promoter (sboA). When the meat is still fresh, the P<i>wap</i>A will regulate a higher production of pigment 1 compared to the pigment 2 production regulated by P<i>sbo</i>A. The pigment 1 will be produced more than the pigment 2 in the fresh meat condition. When the meat is rotten, P<i>wap</i>A will be down-regulated thus decreasing the production of pigment 1 and the P<i>sbo</i>A will be up-regulated, increasing the production of pigment 2. For example: If the pigment 1 is a yellow pigment (AmilGFP) and the pigment 2 is a blue pigment (AmilCP), a strong yellow color will be produced when the meat is still fresh. When the meat is rotten, the pigment will be more blue because the production of the yellow pigment stopped and the blue pigment will be up-regulated, showing greenish-blue color. <br />
</p><br />
</body><br />
</head><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T17:59:20Z<p>Emeraldo88: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Future Plans</z1><br />
</div><br />
</div><br />
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</head><br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a construct the AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). Pigment 1 is regulated by rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). The construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
When the meat is still fresh, the P<i>wap</i>A will regulate a higher production of pigment 1 compared to the pigment 2 production regulated by P<i>sbo</i>A. The pigment 1 will be produced more than the pigment 2 in the fresh meat condition. When the meat is rotten, P<i>wap</i>A will be down-regulated thus decreasing the production of pigment 1 and the P<i>sbo</i>A will be up-regulated, increasing the production of pigment 2. For example: If the pigment 1 is a yellow pigment (AmilGFP) and the pigment 2 is a blue pigment (AmilCP), a strong yellow color will be produced when the meat is still fresh. When the meat is rotten, the pigment will be more blue because the production of the yellow pigment stopped and the blue pigment will be up-regulated, showing greenish-blue color. <br />
</p><br />
</body><br />
</head><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T16:37:43Z<p>Emeraldo88: </p>
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<html><br />
<head><br />
<style><br />
<br />
body { <br />
font-family: Helvetica, Arial, sans-serif;<br />
<br />
}<br />
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color:white;<br />
}<br />
.ctd {<br />
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text-align: center;<br />
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<div class="cte"><br />
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<z1>Future Plans</z1><br />
</div><br />
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</head><br />
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<br />
<br />
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<head><br />
<style><br />
p.margin<br />
{<br />
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margin-left:150px;<br />
margin-right:150px;<br />
font-size:10pt;<br />
line-height:14pt;<br />
color:white;<br />
}<br />
</style><br />
<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a construct the AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). Pigment 1 is regulated by rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). The construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
When the meat is still fresh, the P<i>wap</i>A will not be down-regulated and the P<i>sbo</i>A will not be up-regulated. The pigment 1 and pigment 2 will be produced at the same time. When the meat is rotten, P<i>wap</i>A will be down-regulated thus decreasing the production of pigment 1 and the P<i>sbo</i>A will be up-regulated, increasing the production of pigment 2. For example: If the pigment 1 is a yellow pigment (AmilGFP) and the pigment 2 is a blue pigment (AmilCP), a green pigment (combination of yellow and blue) will be produced when the meat is still fresh. When the meat is rotten, the pigment will be more blue because the production of the yellow pigment stopped and the blue pigment will be up-regulated. <br />
</p><br />
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{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T16:26:22Z<p>Emeraldo88: </p>
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<z1>Future Plans</z1><br />
</div><br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a construct the AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). Pigment 1 is regulated by rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). The construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br><br />
<p class="margin"><br />
When the meat is still fresh, the P<i>wap</i>A will not be down-regulated and the P<i>sbo</i>A will not be up-regulated. The pigment 1 and pigment 2 will be produced at the same time. When the meat is rotten, P<i>wap</i>A will be down-regulated thus decreasing the production of pigment 1 and the P<i>sbo</i>A will be up-regulated, increasing the production of pigment 2. <br />
</p><br />
</body><br />
</head><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T16:01:19Z<p>Emeraldo88: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Future Plans</z1><br />
</div><br />
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</head><br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a construct that placed AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). Pigment 1 is regulated by rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). The construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=450 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=750 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<br />
</body><br />
</head><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/File:Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.pngFile:Groningen2012 EJ 20120924 downregulated promoter-upregulated promoter.png2012-10-26T16:00:45Z<p>Emeraldo88: uploaded a new version of &quot;File:Groningen2012 EJ 20120924 downregulated promoter-upregulated promoter.png&quot;</p>
<hr />
<div></div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T15:58:09Z<p>Emeraldo88: </p>
<hr />
<div>{{HeaderGroningen2012}}<br />
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<style><br />
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font-family: Helvetica, Arial, sans-serif;<br />
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<br><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Future Plans</z1><br />
</div><br />
</div><br />
</body><br />
</head><br />
</html><br />
<br />
<br />
<html><br />
<head><br />
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font-size:10pt;<br />
line-height:14pt;<br />
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</style><br />
<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
<z2><i>Update! (26th October 2012)</i></z2><br><br><br />
After the European regional jamboree, we succeeded to make a construct that placed AmilGFP under regulation of P<i>wap</i>A (rotten meat down-regulated promoter). Pigment 1 is regulated by rotten meat down-regulated promoter (wapA) and pigment 2 is regulated by the up-regulated promoter (sboA). The construct is a pilot construct for the following diagram:<br><br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/8/85/Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>hover you mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<br />
</body><br />
</head><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/File:Groningen2012_EJ_20120924_downregulated_promoter-upregulated_promoter.pngFile:Groningen2012 EJ 20120924 downregulated promoter-upregulated promoter.png2012-10-26T15:46:10Z<p>Emeraldo88: </p>
<hr />
<div></div>Emeraldo88http://2012.igem.org/Team:Groningen/in_developmentTeam:Groningen/in development2012-10-26T13:54:51Z<p>Emeraldo88: </p>
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<z1>Future Plans</z1><br />
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<p class="margin"><br><br />
We proved the principle of our Food Warden system by developing a construct that enables <i>Bacillus subtilis</i> to form a pigment as a reaction to rotten meat. We designed different constructs which can improve the function of the Food Warden by tuning the pigment production or turning the Food Warden into a multi-color system.<br />
<br><br><br><br />
<z2>Tuning system of Pigment Production</z2><br />
<br><br><br />
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 to produce a system that is truly more accurate and reliable than the best-before date. The tuning of this device requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:<br><br />
<ul class="indentlist"><br />
<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><br />
<font color=#FF6700><b>2. </b></font>Degree of spoilage health risk: The undefined nature of current assessments of spoiling degrees in food (see <a href="https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative"><FONT COLOR=#ff6700>Stop the food waste initiative</font></a>) make this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of this standard for specific types of meat.<br><br />
<font color=#FF6700><b>3. </b></font>Pigment production: The control of pigment production dynamics will depend on the outcome 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. <br />
</ul></p><br />
<br><br />
<p class="margin"><br />
The pigment production can be tuned to a desired speed and sensitivity with different regulating promoters, different rbs and with a 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 is shown below:<br></p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br><br />
<p class="margin"><br />
When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is designed in a way 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). <br />
<br><br><br><br />
<z2>Multi-colored Pigment System</z2><br />
<br><br><br />
The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this system 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><br />
<ul class="indentlist"><br />
<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. <br />
<br><br />
<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.<br />
<br><br />
<font color=#FF6700><b>3. </b></font>Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that is contaminated with another organism.<br />
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.<br />
<br><br />
</ul><br />
</p><br />
<p class="margin"><br />
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. <br />
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 options were considered:<br><br />
<ul class="indentlist"><br />
<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><br />
<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><br />
<br />
<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 (go to the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><font color=#FF6700>Sensor page</font></A> for more information on the downregulated genes and operons).<br><br />
<br />
<font color=#FF6700><b>4. </b></font>Including an operator for the transcription of the control pigment that allows repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.<br><br />
The construct can be put as following:<br><br />
<br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/c/cf/Groningen2012_EJ_20120924_negative_feedback_-_repression_system.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
<font color=#FF6700><b>5. </b></font>Placing a control pigment that reacts to a 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><br />
The construct can be put as following:<br><br><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/7/7a/Groningen2012_EJ_20120924_pigment-pigment_reaction-repressed.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
When the rotten meat promoter 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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" width=400 /><img src="https://static.igem.org/mediawiki/2012/6/65/Groningen2012_EJ_20120924_ctrl_pigment_with_second_compound_with_positive_feedback_construct.png" class="preview" width=700 /></a><br />
</li><br />
</ul><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</div><br><br />
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.<br />
</ul></p><br />
<br><br />
<p class="margin"><br />
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. <br />
<br><br><br><br />
</p><br />
</body><br />
</head><br />
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<br />
{{Template:SponsorsGroningen2012}}</div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T13:46:52Z<p>Emeraldo88: </p>
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update! (26th October 2012)</i></z3><br><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. We engineered the construct inside Psaccm backbone (BBa_K818000)<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"></img><br />
<br><br />
<p class=caption><i>AmilGFP under regulation of PwapA</i><br />
</p><br />
</div><br />
<br />
<p class="margin"><br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A 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 P<i>sbo</i>A in the same situation. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
</p><br />
<br />
</body><br />
</head><br />
</html><br />
<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div></A><br />
<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T13:46:16Z<p>Emeraldo88: </p>
<hr />
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Construct</z1><br />
</div><br />
</div><br />
</body><br />
</head><br />
</html><br />
<br />
<br />
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}<br />
</style><br />
<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update! (26th October 2012)</i></z3><br><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. We engineered the construct inside Psaccm backbone (BBa_K818000)<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"></img><br />
<br><br />
<p class=caption><i>AmilGFP under regulation of PwapA<br />
</p><br />
</div><br />
<br />
<p class="margin"><br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A 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 P<i>sbo</i>A in the same situation. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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{{Template:SponsorsGroningen2012}}<br />
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<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div></A><br />
<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T13:25:18Z<p>Emeraldo88: </p>
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<z1>Construct</z1><br />
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update! (26th October 2012)</i></z3><br><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></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="165"></img><br />
<br><br />
<p class=caption>caption!<br />
</p><br />
</div><br />
<br />
<p class="margin"><br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A 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 P<i>sbo</i>A in the same situation. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T13:24:45Z<p>Emeraldo88: </p>
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update! (26th October 2012)</i></z3><br><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><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="165"></img><br />
<br><br />
<p class=caption>caption!<br />
</p><br />
</div><br />
<br />
<br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A 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 P<i>sbo</i>A in the same situation. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
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<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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</html></div>Emeraldo88http://2012.igem.org/File:Pwapa_construct_amilgfp.pngFile:Pwapa construct amilgfp.png2012-10-26T13:23:01Z<p>Emeraldo88: </p>
<hr />
<div></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T13:07:54Z<p>Emeraldo88: </p>
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update! (26th October 2012)</i></z3><br><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><br />
<br />
<br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A 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 P<i>sbo</i>A in the same situation. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
</p><br />
<br />
</body><br />
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<br />
{{Template:SponsorsGroningen2012}}<br />
<br />
<html><br />
<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div></A><br />
<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T12:59:29Z<p>Emeraldo88: </p>
<hr />
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<z1>Construct</z1><br />
</div><br />
</div><br />
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<br />
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</style><br />
<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update! (26th October 2012)</i></z3><br><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 />
<br />
<br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A in the presence of fresh meat but not in the rotten meat. This proved that our <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T12:57:02Z<p>Emeraldo88: </p>
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<z1>Construct</z1><br />
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
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<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update!</i></z3><br><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 />
<br />
<br />
We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A, in the presence of fresh meat and rotten meat. The AmilGFP was produced under regulation of P<i>wap</i>A in the presence of fresh meat but not in the rotten meat. This proved that our <br />
<br />
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<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
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</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T12:50:46Z<p>Emeraldo88: </p>
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<z1>Construct</z1><br />
</div><br />
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update!</i></z3><br><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. We used AmilGFP to test our new rotten meat down-regulated promoter compared to the production of the pigment under the up-regulated promoter, P<i>sbo</i>A in the presence of fresh meat and rotten meat, as the proof of principle of our construct idea. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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{{Template:SponsorsGroningen2012}}<br />
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<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
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<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T12:46:29Z<p>Emeraldo88: </p>
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<z1>Construct</z1><br />
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update!</i></z3><br><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. We used AmilGFP to test our new promoter to compare the production of the pigment in the presence of fresh meat and rotten meat, as the proff of principle of our construct idea. <br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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{{Template:SponsorsGroningen2012}}<br />
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<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"><br />
</div></A><br />
<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T12:36:05Z<p>Emeraldo88: </p>
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<z1>Construct</z1><br />
</div><br />
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update!</i></z3><br><br><br />
After the European regional jamboree, we were back in the lab to build our 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>wapA</i><br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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{{Template:SponsorsGroningen2012}}<br />
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<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
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</div></A><br />
<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/ConstructTeam:Groningen/Construct2012-10-26T12:27:20Z<p>Emeraldo88: </p>
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<z1>Construct</z1><br />
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<p class="margin"><br />
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter. When <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 placed pigment genes under the control of the promoter so that the pigment would be produced when the promoter is activated.<br><br />
</p><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i></p><br />
</li><br />
</ul><br />
</div><br />
<br><br />
<p class="margin"><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><br><br />
<br />
<z3><i>Update!</i></z3><br><br />
After the European regional jamboree, we were back in the lab to build our constructs in the <A HREF="https://2012.igem.org/Team:Groningen/in_development"><FONT COLOR=#ff6700>development page</FONT></A><br />
<br />
</p><br />
<div class="cte"><br />
<div class="ctd"><br />
<z1>Characterization</z1><br />
</div><br />
</div><br />
<br><br />
<p class="margin"><br />
<z2>SboA-AmilGFP</z2><br />
<br><br><br />
1.) <z3>Expression in <i>E. coli</i></z3><br><br />
<i>SboA-AmilGFP</i> 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.<br><br />
<div align="center"><br />
<img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"></img> <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"></img><br />
<br><br />
<p class=caption><i><br />
Left: Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br><br />
Right: Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5α. B3 is SboA-AmilGFP.<br></i><br />
</p><br />
</div><br />
<br><br><br />
<p class="margin"><br />
2) <z3>Expression in <i>B. subtilis</i></z3><br />
<br><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 <z4>spoiled meat</z4> and <z4>without meat</z4>. 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.<br><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="400"></img><br />
<p class=caption><i>Left picture, 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><br />
Right picture: Pelleted cells after 16 hour growth with/without spoiled meat. </i></p><br />
<br><br />
<p class="margin"><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.<br><br><br />
<br />
<img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"></img> <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"></img><br />
<p class=caption><i>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><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.<br></i></p><br />
<br><br><br />
<p class="margin"><br />
<z4>AmilGFP</z4> and <z4>AmilCP</z4> both are <z4>fluorescent proteins</z4>. 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 <z4>strong <i>Bacillus subtilis</i> promoter rrnB</z4>. 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.<br />
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><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><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><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br><br />
<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.</p><br />
</li><br />
</ul><br />
</div><br />
<div align="center"><br />
<ul class="hoverbox"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" width=400 height=257 /><img src="https://static.igem.org/mediawiki/2012/a/ac/Groningen2012_color_over_time.PNG" class="preview" width=700 height=450 /></a><br />
<p class=caption><i>Hover your mouse over the image to see a bigger version!</i><br>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).</p><br />
</li><br />
</ul><br />
</div><br />
<p class="margin"><br />
<z2>SboA-AmilCP</z2><br />
<br><br><br />
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 5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.<br />
<br />
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. See the <A HREF="https://2012.igem.org/Team:Groningen/Sticker"><FONT COLOR=#ff6700>sticker page</FONT></A> for more information.<br />
<br><br />
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{{Template:SponsorsGroningen2012}}<br />
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<A HREF="https://2012.igem.org/Team:Groninge/Kill_Switch" ><div style="position:absolute; right: 0px; bottom: 660px;"><br />
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</div></A><br />
<div style="position:absolute; right: 10px; bottom: 600px;"><br />
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"><br />
</div><br />
</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:53:32Z<p>Emeraldo88: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
</div><br />
<p class=caption><i>Results of TAMC counting. TAMC (colony forming units/gram meat, y axis) of meat incubated at room temperature for indicated time (x axis). Red area indicates the critical values where the meat is not allowed to be distributed for consumption according to the EU.</i></p><br />
<br><br />
<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
<br><br><br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
</div><br />
<p class=caption><i>Smelling test. Minced meat was left at room temperature for 6 hours. The “nastiness” of the meat smell according to the tester was written down.</i></p><br><br />
<p class="margin"><br />
Now that we defined and smelled rotten meat, we want to know what the volatiles are. The rotting of the meat is caused by bacteria proliferating in the meat. These bacteria mix produce a lot of volatiles and we can only smell a few. But what are volatiles? To answer this question we analyzed the rotten meat volatiles with GC-MS.</p><br />
<br><br><br />
<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="500"><br />
</td><br />
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</table><br><br><br />
<br />
<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
<br><br />
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<td width="175px" align="central"><br />
<img src="<br />
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</td><br />
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<z5>Sample bottles with rotten minced meat</z5><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/7/73/Groningen2012_EH_20120727_P7270583.JPG" width="500"><br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
<br />
<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
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<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:45:21Z<p>Emeraldo88: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
</div><br />
<p class=caption><i>Results of TAMC counting. TAMC (colony forming units/gram meat, y axis) of meat incubated at room temperature for indicated time (x axis). Red area indicates the critical values where the meat is not allowed to be distributed for consumption according to the EU.</i></p><br />
<br><br />
<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
<br><br><br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
</div><br />
<p class=caption><i>Smelling test. Minced meat was left at room temperature for 6 hours. The “nastiness” of the meat smell according to the tester was written down.</i></p><br />
<br><br><br />
<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="500"><br />
</td><br />
</tr><br />
</table><br><br><br />
<br />
<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
<br><br />
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<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/8/87/Groningen2012_EH_20120727_P7270580.JPG" width="500"><br />
</td><br />
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</table><br><br><br />
<z5>Sample bottles with rotten minced meat</z5><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/7/73/Groningen2012_EH_20120727_P7270583.JPG" width="500"><br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
<br />
<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
<ul class="hoverboxL"><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:41:59Z<p>Emeraldo88: </p>
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<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="http://1.1.1.3/bmi/2012.igem.org/wiki/images/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
</div><br />
<p class=caption><i>Results of TAMC counting. TAMC (colony forming units/gram meat, y axis) of meat incubated at room temperature for indicated time (x axis). Red area indicates the critical values where the meat is not allowed to be distributed for consumption according to the EU.</i></p><br />
<br><br />
<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
<br><br><br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
</div><br />
<p class=caption><i>Smelling test. Minced meat was left at room temperature for 6 hours. The “nastiness” of the meat smell according to the tester was written down.</i></p><br />
<br><br><br />
<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="500"><br />
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<br />
<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
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<z5>Sample bottles with rotten minced meat</z5><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
<br />
<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
<ul class="hoverboxL"><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<br />
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<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /></a><br />
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<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:37:38Z<p>Emeraldo88: </p>
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<div class="cte"><br />
<div class="ctd"><br />
<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="http://1.1.1.3/bmi/2012.igem.org/wiki/images/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
<br><br><br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
</div><br />
<br><br><br />
<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="500"><br />
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</table><br><br><br />
<br />
<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
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</table><br><br><br />
<z5>Sample bottles with rotten minced meat</z5><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
<br />
<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" class="preview" width=800 /></a><br />
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<ul class=hoverboxR><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" class="preview" width=800 /></a><br />
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</ul><br />
<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
</li><br />
</ul><br />
<ul class=hoverboxM><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<ul class=hoverboxR><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /></a><br />
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</ul><br />
<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:36:59Z<p>Emeraldo88: </p>
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<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="http://1.1.1.3/bmi/2012.igem.org/wiki/images/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
</div><br />
<br><br><br />
<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
<br><br><br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
</div><br />
<br><br><br />
<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="500"><br />
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<br />
<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
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<td width="175px" align="central"><br />
<img src="<br />
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<z5>Sample bottles with rotten minced meat</z5><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
<br />
<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" class="preview" width=800 /></a><br />
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<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /></a><br />
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<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:35:31Z<p>Emeraldo88: </p>
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<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="http://1.1.1.3/bmi/2012.igem.org/wiki/images/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
</div><br />
<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
<br><br><br />
</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
</div><br />
<br><br><br />
<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="https://static.igem.org/mediawiki/2012/4/4c/Groningen2012_EH_20120727_P7270578.JPG" width="500"><br />
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<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
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<td width="175px" align="central"><br />
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<z5>Sample bottles with rotten minced meat</z5><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
<br />
<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
<br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/5/52/Groningen2012_AO_20120924_HP-1_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
</td><br />
<td width="175px" align="central"><br />
<img src="<br />
https://static.igem.org/mediawiki/2012/0/01/Groningen2012_AO_20120924_HP-5_substratestable.png" width="700"><br />
</td><br />
</tr><br />
</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" class="preview" width=800 /></a><br />
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<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /></a><br />
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<br />
<br />
</p><br />
<br />
<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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</html></div>Emeraldo88http://2012.igem.org/Team:Groningen/volatilesTeam:Groningen/volatiles2012-09-27T01:33:58Z<p>Emeraldo88: </p>
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<z1 >Volatiles</z1><br />
</div><br />
</div><br />
<br><br><br />
<z2>When is meat rotten?</z2><br />
<br><br><br />
<div align="center"><br />
<img src="http://1.1.1.3/bmi/2012.igem.org/wiki/images/f/f6/RR_20120807_TAMCpic.jpg"></img><br />
</div><br />
<br><br />
<p class="margin"><br />
To make a rotting sensor, we have to have a definition of rotten meat. For this, we used the guidelines of the European Union (2006, source <a href="http://www.imik.org/wettelijke_context/Europese_hygienerichtlijn_en_microbiologische_criteria.pdf" target=_blank"><font color=#ff6700>(in Dutch)</font></a>) and did a simple Total Aerobic Microbial Count test. With this test, one can estimate the amount of colony forming units (CFU) per gram meat. Also see our food safety page for the is question. Our meat of choice was 70% pork, 30 % beef minced meat from our local supermarket. This type of meat is often bought in too large amounts and leftovers will be restored in the fridge, making it the ideal candidate for our Food Warden system. Minced meat is also easy to handle when it is placed in a jar, so easy for lab work. Most importantly, as a meat lover it is hard to sacrifice a very nice expansive steak for science. We incubated the meat in closed airtight jars, in portions of 1 gram at room temperature, and tested the TAMC at time points 0, 3, 5, 7 and 24 hours. The test has been done in triplo.<br><br></p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/3/34/RR_20120927_TAMCgraph.PNG"></img><br />
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<p class="margin"><br />
To see the working of our own inbuilt rotting sensor, Elbrich bravely tested the smell and appearance of the meat for 5 hours. According to these tests, we humans smell bad meat pretty well too. Side note: the meat has been exposed to air many times so it could be smelled. The color of the meat changed a bit: it turned a bit greyer. <br />
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<img src="https://static.igem.org/mediawiki/2012/0/06/Groningen_RR_20120806_smel_view.jpg" width="500"></img><br />
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<z2>Bad meat volatiles</z2><br />
<br />
<p class="margin"><br />
With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out the exact compounds that influence the behavior of our identified <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>, but we were surprised by what we found…<br><br />
<br><br />
The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data can be obtained. But we discovered that the data were hard to analyze due to the large diversity of the volatiles present. <br />
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<td width="175px" align="central"><br />
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<z5>Picture: Arjan and Tom at work with the GC-MS.</z5><br><br />
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<z5>Sample bottles with rotten minced meat</z5><br><br><br />
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<z5>Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)</z5><br><br><br />
</p><br />
<br><br />
<z2>Introduction to the GC-MS technique</z2><br><br />
<p class="margin"><br />
Substrates in the gas chromatograph are separated by the amount of time needed to pass through a capillary column in the machine. The volatiles, in a gas phase, pass through the column which has a liquid carrier. Because of the constant flow, the equilibrium between the gas phase and a interaction with the liquid carrier is constantly redefined. Interaction with the carrier will slow down the substrate that is traveling through the column. For the HP-1 and HP-5 columns, used in our experimental setup, the interaction between substrate and carrier is based on the boiling point of the substrate. Each substrates has an unique amount of interactions with the carrier and thus a different amount of time is needed to pass through the column. Based on this principle our rotted meat volatiles are separated and later identified with mass spectrometry<br><br> We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat <a href="https://2012.igem.org/Team:Groningen/Sensor" target=_blank"><font color=#ff6700>sensors</font></a>.<br><br />
<br><br />
As stated we used the HP-5 and HP-1 for GC experiments. There is a broad range of columns commercially available, and all are capable of separating substrates bases on different properties. The HP-1 and HP-5 are good columns for general use. Most GC setups at the RUG utilize these columns, but it has to be noted that these columns cannot identify amino compounds. Because of this we will miss some of the volatiles in the rotted meat and unfortunately we did not have the budget to buy more specific columns.<br><br />
<br><br />
HP-5:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091J-433<br><br />
stationary phase: (5%-Phenyl)-95%methylpolysiloxane<br><br />
<br><br />
HP-1:<br><br />
dimensions: 30 m x 0,25 mm x 0,25 um<br><br />
manufactory: Agilent<br> <br />
Column number: 19091Z-433<br><br />
stationary phase: 100% polysiloxane<br><br />
<br><br />
<br><br />
After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the saparated substrates in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of substrates, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.<br><br />
<br> <br />
The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.<br><br />
<br><br />
The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporated and be extracted from the bottle, before being injected into the GC machine. <br />
<br><br />
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.<br><br />
<br><br />
</p><br />
<br />
<z2>Liquid injection with organic solvents</z2><br><br />
<p class="margin"><br />
The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.<br><br />
<br> <br />
For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.<br><br />
<br> <br />
</p><br />
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<z2>Results</z2><br><br />
<p class="margin"><br />
Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.<br><br />
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<td width="175px" align="central"><br />
<img src="<br />
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<td width="175px" align="central"><br />
<img src="<br />
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</table><br><br><br />
We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.<br><br />
<br><br />
He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable. This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.<br><br />
<br> <br />
Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.<br><br />
<br> <br />
Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.<br><br />
<br> <br />
Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.<br><br />
<br><br />
<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/8/85/Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/6/6e/Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_Hexane.png" class="preview" width=800 /></a><br />
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<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/d/d3/Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP5_Data_MeOH.png" class="preview" width=800 /></a><br />
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<z5 style="margin-left:150px;">GC-MS spectras using HP-5 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br />
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<ul class="hoverboxL"><br />
<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/ec/Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_CH2Cl2.png" class="preview" width=800 /></a><br />
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<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/e/e9/Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_Hexane.png" class="preview" width=800 /></a><br />
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<li><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" width=230 /><img src="https://static.igem.org/mediawiki/2012/thumb/0/0b/Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png/800px-Groningen2012_ID_JP20120925_GC_HP1_Data_MeOH.png" class="preview" width=800 /></a><br />
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</p><br />
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<br><br><br><br><br><br><br><br />
<z5 style="margin-left:150px;">GC-MS spectras using HP-1 column and the organic solvents: hexane, dichloromethane and methanol.</z5><br><br><br />
<z2>Future plans</z2><br><br />
<p class="margin"><br />
If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of <i>B. subtilis</i> with our construct.<br><br><br />
</p><br />
<br />
<z2>Reference and acknowledgement</z2><br><br />
<p class="margin"> <br />
We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.<br><Br></p><br />
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