"
Team:Groningen/Construct
From 2012.igem.org
m |
|||
| Line 387: | Line 387: | ||
<html> | <html> | ||
<a href="https://2012.igem.org/Team:Groningen/Kill_Switch"> | <a href="https://2012.igem.org/Team:Groningen/Kill_Switch"> | ||
| - | <div style="position:absolute; | + | <div style="position:absolute; right: 0px; bottom: 760px;"> |
<img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"> | <img src="https://static.igem.org/mediawiki/2012/2/22/Groningen2012_RR_20120910_nextstage.png" width="150"> | ||
</div> | </div> | ||
</a> | </a> | ||
| - | <div style="position:absolute; | + | <div style="position:absolute; right: 10px; bottom: 700px;"> |
<img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"> | <img src="https://static.igem.org/mediawiki/2012/8/87/Groningen2012_RR_20120910_orangearrow.png"> | ||
</div> | </div> | ||
</html> | </html> | ||
Revision as of 21:22, 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.
|
|
|
Hover your mouse over the image to see a bigger version! |
We use our Bacillus subtilis backbone (BBa_K818000) that has sacA and a chloramphenicol resistance gene for chromosomal integration
and antibiotic screening of transformants respectively. This backbone also has E. coli origin of replication, so it can be amplified inside
E. coli.
After the European regional jamboree, we were back in the lab to build our planned constructs in the
development page.
We coupled PwapA, a promoter that was down-regulated by the presence of rotten meat volatiles, with amilGFP coding gene.
We engineered the construct inside psac-cm backbone (BBa_K818000)
|
|
AmilGFP under regulation of PwapA |
The pigment production activity of PwapA-amilGFP was compared with the production of the pigment regulated by the up-regulated promoter (PsboA) in the presence of fresh meat and rotten meat. The yellow colour was produced under regulation of PwapA in the presence of fresh meat but absent in the presence of rotten meat.
SboA-AmilGFP is strongly expressed in E. coli, on plate and in liquid culture, at normal growth conditions. On plate,
the yellow color is less visible compared to the cell pellet in liquid culture.
|
(left) Pellet of SboA-AmilGFP in E. coli DH5a.
(right) Plate with SboA connected to several pigment genes inside E. coli DH5a. B3 is SboA-AmilGFP.
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
|
(left) From left to (right) Wildtype grown without meat, B.subtilis(sboA-AmilGFP) grown without meat, Wildtype grown with spoiled meat, B.subtilis(sboA-AmilGFP) grown with spoiled meat, two jars of spoiled meat.
(right) Pelleted cells after 16 hour growth with/without spoiled meat.
To check whether the difference in color was not the result of the promoter activation by the presence of meat in general, we also compared
the growth of B. subtilis sboA-AmilGFP strain subjected to fresh meat and rotten meat. We grew the strain in Luria Broth in the microarray
setup for 12 hours and measured OD (600 nm), absorbance (395 nm) and assayed the color of the cells when pelleted. Below you can see the results:
while grown without meat volatiles and with fresh meat volatiles, our device strain still produces yellow color. The color was produced faster
and in a larger amount when the device strain was subjected to volatiles from spoiling meat.
|
(left) Absorption of AmilGFP (395 nm) per amount of cells (OD(600)) of Bacillus subtilis sboA-AmilGFP strain grown for 12 hours while subjected to spoiled meat, fresh meat, or no meat.
(right) Visibility of yellow color of pelleted cells by eye. Assay done with 5 previously made pellets of different color intensities as a reference to ensure objectivity.
|
|
Hover your mouse over the image to see a bigger version!
Bacillus subtilis, 1000x, AmilGFP fluorescence measurement, exposure time = 50 ms, ex = 470 nm, em = 514 nm. Clockwise, from the top (left) 1) positive control: strong promoter rrnB with AmilGFP. 2) SboA-AmilGFP exposed to spoiled meat. 3)Wild type 4)SboA-AmilGFP grown without meat.
|
|
Hover your mouse over the image to see a bigger version!
Color of pellets of sboA-GFP in a no-meat environment (above) and exposed to spoiled meat (below)after 6 hours(6H), 8 hours (8H), 10 hours (10H), and 12 hours (12H).
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.
Our sponsors:
