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From 2012.igem.org

We wanted to use pigments as reporter in our designed genetic construct. Why did we choose for pigments and not use a common reporter like Green Fluorescent Protein (GFP)? Well, we imagined our Food Warden system to become a product for the consumer. Then it is not very likely to expect that everybody has a fluorescence microscope available at home. The advantage of pigments is that they are recognizable by the naked eye and come in many color variaties, but a main challenge we had to execpt is that these pigments were never introducted to Bacillus subtilis before. We decided to take the risk, and look what interesting results we got!

We identified many pigments in the Registry and choose three pigment BioBricks that we could express independently: so we could create different colors for every single device. The parts that have been utilized as a reporter gene are:

1. Lycopene (BBa_K274100)
   Coral red pigment biobrick CrtEBI with RBS (BBa_K274100), created by Team Cambridge 2009, was used as one of our reporter gene in our volatile detection device. Details concerning this part can be found in Team Cambridge 2009 page.BBa_K274100.
   
   Our team has managed to couple this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K823023 (plasmid backbone for B. subtilis, engineered by team LMU Munich 2012), to allow color expression in B. subtilis. We utilized a strong RBS BBa_B0034 for pigment expression in E. coli and B. subtilis.
   Picture: E. coli DH5α containing fnr promoter + lycopene coding gene. The red color was expressed due to the leakiness of the fnr promoter.
   
   Picture: B. subtilis 168 containing promoters (alsT, fnr, and sboA) coupled with the lycopene coding gene. The red color was not as highly expressed as in E. coli. This phenotype was due to the weak promoter of B. subtilis that was used in this construct and the RBS for E. coli that was utilized for this device construct.



2. AmilCP (BBa_K592009)
   AmilCP is a blue/purple chromoprotein biobrick part (BBa_K592009), created by Team Uppsala Sweden 2011, and was used as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. BBa_K592009. AmilCP has a sequence similar to the other coral chromoprotein, only its maximum absorption is shifted by 10 nm causing the color to appear blue instead of purple.
   
   Our team has managed to couple this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 (plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in B. subtilis. We utilized a strong RBS BBa_B0034 for pigment expression in E. coli and B. subtilis. This reporter coding gene was strongly expressed in E. coli due to the leakiness of the promoters. However, the expression in B. subtilis was not as leaky as in E. coli. This phenotype in B. subtilis was due to the promoter strength and the RBS that was used in this construct. The promoters are considered as weak promoters, therefore induction by volatile that is produced by the rotten meat is needed for color expression.
   
   
   
   
   Picture: E. coli DH5α containing sboA promoter + amilCP (upper picture). Blue/purple colour was highly expressed due to the leakiness of the promoter and a strong RBS for E. coli. However, the expression of this part in B. subtilis was more subtle. B. subtilis containing sboA promoter + amilCP (bottom picture) displayed faint blue colour on its colony.



3. AmilGFP (BBa_K592010)
   AmilGFP is a yellow chromoprotein biobrick part (BBa_K592010), created by Team Uppsala Sweden 2011, and was used as one of the reporter genes in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. BBa_K592010. This chromoprotein is part of the green chromoprotein with maximum absorbtion at 503 nm.
   
   Our team has managed to couple this biobrick part with our promoters: alsT, fnr, and sboA. The cloning was done in BBa_K818000 (plasmid backbone for B. subtilis, engineered by team Groningen 2012), to allow color expression in B. subtilis. We utilized a strong RBS BBa_B0034 for pigment expression in both E. coli and B. subtilis. This reporter coding gene was strongly expressed in E. coli due to the leakiness of the promoters, while the expression in B. subtilis without induction was considerably low. This phenotype in B. subtilis was due to the promoter strength and the RBS that was used in this construct. The promoters are considered as weak promoters, therefore induction by volatiles from the rotten meat is needed for strong color expression.
   
   The volatile detection device containing sboA promoter and amilGFP has been tested in the presence of the rotten and fresh meat. We found that under the presence of rotten meat our volatile detection device produced bright yellow color. On the other hand, our device that was exposed to the volatiles produced by the fresh meat, did only produce a small amount of yellow color. The comparison between B. subtilis containing sboA + amilGFP to B. subtilis 168 wildtype under the presence of rotten meat volatiles resulted in yellow pigment production by our device while no yellow pigment was produced in the wildtype strain. The yellow color produced by our device is strong enough to observe by the human naked eye.
   
   
   
   
   Picture: E. coli DH5α containing sboA promoter + amilGFP. Yellow color was highly expressed due to the leakiness of the promoter and a strong RBS for E. coli.
   
   
   
   
   
   Picture: Our setup for the volatile detection experiment using the sboA+amilGFP device (upper picture). We compared the pigment expression by our device with the wildtype strain in the presence of rotten meat and without the presence of meat. When our device sensed volatiles produced by the rotten meat, the promoter was induced, resulting in the activation of the amilGFP coding gene and the production of the yellow color. At the end of the experiment, the cells were harvested and spun down to obtain a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatiles exhibited strong yellow colour while the wildtype strain and the device that was not exposed to the volatiles were white (bottom picture).