Team:Groningen/Construct
From 2012.igem.org
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.
To further characterize the difference in amilGFP expression under the PsboA promoter, we measured the fluorescence of amilGFP (ex = 470 nm, em = 514 nm) by flow cytometry. We let our strain grow in the presence of spoiled and fresh meat for nine hours. As showed in the figures below, a clear difference in fluorescence can be seen after six hours of incubation.
We observed that the fluorescence intensity is also slightly influenced by the growth speed of the bacterium: the slower growing culture “fresh 2” (see pictures B and C) has a lower expression of amilGFP compared to the faster growing culture “fresh 1”. However, this difference can be neglected when compared to the difference of the cultures subjected to fresh and spoiled meat while having the same growth speed. This confirmed the importance of finding ways to control the growth of the bacterium inside our sticker by modeling.