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Team:Groningen/OurBiobrick
From 2012.igem.org
These are the biobricks that team Groningen submitted to the registry.
| <groupparts>iGEM012 Groningen</groupparts> |
This backbone was designed to fulfill the need of a working Bacillus subtilis backbone for our project. This backbone plasmid was derived from pSac-Cm by insertion of the biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences and the sequence for red fluorescent protein (RFP) in its multiple cloning site (MCS).
This backbone has a multi host replication origin and replicates in both E. coli and Bacillus subtilis. The plasmid is designed to integrate a cloned insert into the B. subtilis chromosome via double recombination between plasmid and chromosomal sacA sequences. This makes it easy to check for double crossover problems after transformation in Bacillus subtilis: transformants with the correct insertion will not be able to metabolize sucrose.
Another advantage of using this plasmid is that it gives a stable, single copy plasmid integration inside the Bacillus subtilis chromosome, therefore, antibiotic selection is not necessary once the insert is transformed into Bacillus subtilis. This enables easy, stable cloning.
The terminator insertion after the suffixes in combination of the red fluorescent protein (RFP) were meant to make cloning easier and faster: new biobricks can be inserted into this vector by replacement of the RFP biobrick. E. coli transformants with inserts will not produce red color (as the RFP is replaced by the insert), so the colonies can be picked easily (see the picture below).
The plasmid is transformed into any B. subtilis 168 host with selection for chloramphenicol (cat gene) resistance. This plasmid can be amplified inside E.coli with selection for chloramphenicol or ampicillin, and using red-white screening (see above). As described by the iGEM Groningen 2010 team, the red fluorescent protein cannot be produced in Bacillus subtilis.
SboA and Fnr are the two promoters in Bacillus subtilis that are the most upregulated by the rotten meat's volatiles. See the sensor page for more information on how we identified them, and the construct page for the characterization of these promoters.
The promoter that was initially chosen for the food warden construct was the alsT promoter. This gene is one of several genes that is regulated by TnrA. TnrA is a regulator in B. subtilis that is triggered by the availability of nitrogen sources. It regulates a few genes and one of them that is repressed is alsT. TnrA is only active when the amount of nitrogen is low. Since alsT is repressed by TnrA, we suggested that it gets activated as soon as TnrA is depleted due to the availability of nitrogen (Kayumov et al., 2008). There are a few components in rotting meat that contain nitrogen. One of these components is ammonia, a highly volatile and abundant compound. These volatiles would trigger the depletion of TnrA, removing it from the alsT promoter, activating the meat warden construct.
A coding device to generate reporters (AmilCP, Lycopene, and AmilGFP) under regulation of sboA promoter. When the sboA promoter is activated, the downstream reporter will be produced. Please go to the Construct page for a detailed characterization of the working of the SboA-AmilGFP construct.
A coding device to generate lycopene (red pigment) under regulation of fnr promoter (BBa_K818210) and to generate AmilGFP under regulation of alsT promoter (BBa_K818310). When the promoter is activated, the downstream reporter will be produced.
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