Team:Groningen/Construct
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- | + | <body> | |
- | + | <br> | |
- | + | <div class="cte"> | |
- | .hoverbox | + | <div class="ctd"> |
- | + | <z1>Construct</z1> | |
- | + | </div> | |
- | + | </div> | |
- | + | <p> | |
- | + | <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> | |
- | + | </p> | |
- | + | <table class="centertable"> | |
- | + | <tr> | |
- | { | + | <td align="center"> |
- | + | <ul class="hoverbox"> | |
- | + | <li> | |
- | + | <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> | |
- | + | </li> | |
- | + | </ul> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td align="center"> | |
- | + | <p class="captionnomargin"> | |
- | + | Hover your mouse over the image to see a bigger version! | |
- | + | </p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | </table> | |
- | + | <br> | |
- | + | <p> | |
- | + | 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> | |
- | + | <z2>Update! (26th October 2012)</z2> | |
- | + | <br> | |
- | + | <br> | |
- | + | After the European regional jamboree, we were back in the lab to build our planned constructs in the | |
- | + | <a class="inlink" href="https://2012.igem.org/Team:Groningen/in_development">development page</a>. | |
- | + | We coupled P<i>wap</i>A, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene. | |
- | + | We engineered the construct inside psac-cm backbone (BBa_K818000) | |
- | + | <br> | |
- | + | <br> | |
- | } | + | </p> |
- | + | <table class="centertable"> | |
- | + | <tr> | |
- | + | <td align="center"> | |
- | + | <img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td align="center"> | |
- | + | <p class="captionnomargin"> | |
- | + | AmilGFP under regulation of PwapA | |
- | + | </p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | </table> | |
- | + | <br> | |
- | + | <p> | |
- | + | The pigment production activity of P<i>wap</i>A-amilGFP was compared with the production of the pigment regulated by the up-regulated promoter | |
- | + | (P<i>sbo</i>A) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of P<i>wap</i>A in the presence of | |
- | + | fresh meat but absent in the presence of rotten meat. | |
- | + | </p> | |
- | + | <div class="cte2"> | |
- | + | <div class="ctd2"> | |
- | + | <z1>Characterization</z1> | |
- | + | </div> | |
- | + | </div> | |
- | + | <p> | |
- | </ | + | <br> |
- | <p | + | <z2>SboA-AmilGFP</z2> |
- | 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> |
- | < | + | <z3>Expression in <i>E. coli</i></z3> |
- | <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> | + | <i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, |
- | <p class= | + | the yellow color is less visible compared to the cell pellet in liquid culture. |
- | </ | + | <br> |
- | </ | + | </p> |
- | </ | + | <table class="centertable"> |
- | <br> | + | <tr> |
- | <p | + | <td align="center"> |
- | 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> | + | <img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"> |
- | + | <img src="http://partsregistry.org/wiki/images/e/ed/Groningen2012_AP20120926_ecolisboApigments.jpg" width="400"> | |
- | < | + | </td> |
- | After the European regional jamboree, we were back in the lab to build our planned constructs in the < | + | </tr> |
- | < | + | </table> |
- | <img src="https://static.igem.org/mediawiki/2012/b/bf/Pwapa_construct_amilgfp.png" width="350"></ | + | <p class="caption"> |
- | < | + | (left) Pellet of SboA-AmilGFP in <i>E. coli</i> DH5a. <br> |
- | <p class= | + | (right) Plate with SboA connected to several pigment genes inside <i>E. coli</i> DH5a. B3 is SboA-AmilGFP. |
- | </ | + | </p> |
- | </ | + | <p> |
- | + | <br> | |
- | <p | + | <z3>Expression in <i>B. subtilis</i></z3> |
- | 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> |
- | + | <br> | |
- | </p> | + | sboA-AmilGFP was shown to be very weakly expressed in <i>Bacillus subtilis</i> on LB plate (faint color formation after 2 days). |
- | <div class=" | + | This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in <i>B. subtilis</i> subjected to |
- | <div class=" | + | volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated <i>B. subtilis</i>SboA-AmilGFP and |
- | <z1>Characterization</z1> | + | <i>B. subtilis</i>Wildtype from plate into flasks of Luria Broth subjected to <z5>spoiled meat</z5> and <z5>without meat</z5>. |
- | </div> | + | 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: |
- | </div> | + | <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. |
- | <z2>SboA-AmilGFP</z2> | + | <br> |
- | <br><br> | + | <br> |
- | + | </p> | |
- | <i>SboA-AmilGFP</i> is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate, the yellow | + | <table class="centertable"> |
- | < | + | <tr> |
- | <img src="http://partsregistry.org/wiki/images/thumb/6/6c/Groningen2012_AP20120924_EcoliSboAamilGFP.jpg/200px-Groningen2012_AP20120924_EcoliSboAamilGFP.jpg" width="165"> | + | <td align="center"> |
- | < | + | <img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"> |
- | <p class=caption | + | <img src="http://partsregistry.org/wiki/images/a/ae/Groningen2012_AP20120926_sboAamilGFPsetuppellets.jpg" width="400"> |
- | + | </td> | |
- | + | </tr> | |
- | </p> | + | </table> |
- | < | + | <p class="caption"> |
- | + | (left) From left to (right) Wildtype grown without meat, <i>B.subtilis</i>(sboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, <i>B.subtilis</i>(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.<br> | |
- | + | (right) Pelleted cells after 16 hour growth with/without spoiled meat. | |
- | + | </p> | |
- | <br><br> | + | <p> |
- | 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 < | + | <br> |
- | <img src="http://partsregistry.org/wiki/images/6/66/Groningen2012_AP20120924_sboAamilGFPsetup_small.jpg" width="325"> | + | 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 |
- | <p class=caption> | + | 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: | |
- | <br> | + | 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. | |
- | 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"> | + | </p> |
- | <p class=caption> | + | <table class="centertable"> |
- | + | <tr> | |
- | <br> | + | <td align="center"> |
- | <p | + | <img src="http://partsregistry.org/wiki/images/9/96/Groningen2012_RR_absorbance_vs_time.jpg" width="375"> |
- | < | + | <img src="http://partsregistry.org/wiki/images/4/4c/Groningen2012_RR_growth_in_micarraysetup.png" width="315"> |
- | 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> | + | </td> |
- | < | + | </tr> |
- | <ul class="hoverbox"> | + | </table> |
- | <li> | + | <p class="caption"> |
- | <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> | + | (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> |
- | <p class=caption> | + | (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. |
- | <i>Bacillus subtilis</i>, 1000x, AmilGFP fluorescence measurement, exposure time = 50 ms, ex = 470 nm, em = 514 nm. Clockwise, from the top left | + | </p> |
- | < | + | <br> |
- | < | + | <p> |
- | < | + | <z5>AmilGFP</z5> and <z5>AmilCP</z5> both are <z5>fluorescent proteins</z5>. We decided to quantify the amount of AmilGFP inside our <i>Bacillus subtilis</i> |
- | + | strain when subjected to spoiled meat and without meat. As a positive control, we paired the AmilGFP coding gene to the <z5>strong <i>Bacillus subtilis</i> | |
- | <ul class="hoverbox"> | + | 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 |
- | <li> | + | 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 |
- | <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> | + | was highly upregulated, thus more amilGFP was expressed. |
- | <p class=caption> | + | Previous tests showed that the intensity of AmilGFP expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain that was exposed to fresh meat was the same as |
- | < | + | the intensity of AmilGFP that was expressed by <i>Bacillus subtilis</i> sboA-AmilGFP strain exposed to a no-meat environment. |
- | < | + | <br> |
- | + | </p> | |
- | + | <table class="centertable"> | |
- | <z2>SboA-AmilCP</z2> | + | <tr> |
- | <br><br> | + | <td align="center"> |
- | 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. | + | <ul class="hoverbox"> |
- | + | <li> | |
- | 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="#"> |
- | <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 /> |
- | < | + | <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> | |
- | < | + | </li> |
- | </ | + | </ul> |
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p class="caption"> | ||
+ | Hover your mouse over the image to see a bigger version!<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> | ||
+ | <table class="centertable"> | ||
+ | <tr> | ||
+ | <td align="center"> | ||
+ | <ul class="hoverbox"> | ||
+ | <li> | ||
+ | <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> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p class="caption"> | ||
+ | Hover your mouse over the image to see a bigger version!<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> | ||
+ | <p> | ||
+ | <br> | ||
+ | <z2>SboA-AmilCP</z2> | ||
+ | <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. | ||
+ | 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 class="inlink" href="https://2012.igem.org/Team:Groningen/Sticker">sticker page</a> for more information. | ||
+ | </p> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
+ | </body> | ||
</html> | </html> | ||
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- | < | + | <a href="https://2012.igem.org/Team:Groningen/Kill_Switch"> |
- | <img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"> | + | <div style="position:absolute; (right) 0px; bottom: 760px;"> |
- | </div></ | + | <img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"> |
- | <div style="position:absolute; right | + | </div> |
- | <img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"> | + | </a> |
- | </div> | + | <div style="position:absolute; (right) 10px; bottom: 700px;"> |
+ | <img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"> | ||
+ | </div> | ||
</html> | </html> |
Revision as of 20:59, 26 October 2012
Our construct idea is simple and effective: there will be a production of pigment under the regulation of a rotten-meat reactive promoter.
When Bacillus subtilis senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the
production of downstream genes. We placed pigment genes under the control of the promoter so that the pigment would be produced
when the promoter is activated.
We use our Bacillus subtilis backbone (BBa_K818000) that has sacA and a chloramphenicol resistance gene for chromosomal integration
and antibiotic screening of transformants respectively. This backbone also has E. coli origin of replication, so it can be amplified inside
E. coli.
After the European regional jamboree, we were back in the lab to build our planned constructs in the
development page.
We coupled PwapA, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene.
We engineered the construct inside psac-cm backbone (BBa_K818000)
The pigment production activity of PwapA-amilGFP was compared with the production of the pigment regulated by the up-regulated promoter (PsboA) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of PwapA in the presence of fresh meat but absent in the presence of rotten meat.
SboA-AmilGFP is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate,
the yellow color is less visible compared to the cell pellet in liquid culture.
sboA-AmilGFP was shown to be very weakly expressed in Bacillus subtilis on LB plate (faint color formation after 2 days).
This is probably due to the leakiness of the promoter. We tested the expression of sboA-AmilGFP in B. subtilis subjected to
volatiles from spoiled meat using the same setup as we used for the microarray. Firstly, we inoculated B. subtilisSboA-AmilGFP and
B. subtilisWildtype from plate into flasks of Luria Broth subjected to
To check whether the difference in color was not the result of the promoter activation by the presence of meat in general, we also compared
the growth of B. subtilis sboA-AmilGFP strain subjected to fresh meat and rotten meat. We grew the strain in Luria Broth in the microarray
setup for 12 hours and measured OD (600 nm), absorbance (395 nm) and assayed the color of the cells when pelleted. Below you can see the results:
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.
AmilCP is expressed less strongly in Bacillus subtilis than AmilGFP. On plate, not induced by volatiles, a faint blue-greyish color is visible after
5 days of incubation. In liquid culture, it is not visible without induction by spoiled meat volatiles.
However, after placing Bacillus subtilis in our sticker and exposing the sticker to rotten meat volatiles, it turned into a clear purple color.
See the sticker page for more information.