Team:Groningen/pigmentproduction

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Revision as of 13:23, 25 September 2012




Pigments



We chose three different pigments as our reporter gene in our designed construct. These three parts were used independently to express different colours for every single device. The parts that have been utilized as a reporter gene are as followed:

  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 colour expression in B. subtilis. We utilized 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 colour was expressed due to the leakiness of the fnr promoter.

    Picture: B. subtilis 168 containing promoters (alsT, fnr, and sboA) coupled with lycopene coding gene. The red colour 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 our reporter gene in our volatile detection device. Details concerning this part can be found in Team Uppsala Sweden 2011 page. BBa_K592009. AmilCP has the very similar sequence as the other coral chromoprotein, only its maximum absorption is shifted by 10 nm and causing the colour appear as blue instead of purple to human eyes.

    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 colour expression in B. subtilis. We utilized 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 colour 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 our reporter gene 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 colour expression in B. subtilis. We utilized 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 volatile that is produced by the rotten meat is needed for strong colour 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 colour. On the other hand, our device that was exposed to the volatile produced by the fresh meat, did not produce yellow colour. The comparison between B. subtilis containing sboA + amilGFP to B. subtilis 168 wildtype under the presence of rotten meat volatile was also showed the yellow pigment production by our device while no yellow pigment detected from the wildtype strain. The yellow colour produced by our device are strong enough for the observation by human naked eyes.




    Picture: E. coli DH5α containing sboA promoter + amilGFP. Yellow colour was highly expressed due to the leakiness of the promoter and a strong RBS for E. coli.





    Picture: Our setup for volatile detection experiment using sboA+amilGFP device (upper picture). We compared the pigment expression by our device to the wildtype strain in the presence of rotten meat and without the presence of meat. When our device sense volatile produced by the rotten meat, the promoter was induced, therefore amilGFP coding gene was activated and the yellow colour was produced. In the end of the experiment, the cells were harvested and spun down to get a better view on the cell pellet. The cell pellet from our device that was exposed to the rotten meat volatile exhibited strong yellow colour while the wildtype strain and the device that was not exposed to the volatile were white (bottom picture).



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