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Team:Groningen/OurBiobrick
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Revision as of 12:06, 26 September 2012
This are the biobricks that our team 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 biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences, and red fluorescent protein sequence (RFP) in between the prefix and suffix in its multiple cloning sites (MCS).
This backbone has a multi host replication origin and replicates in 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 and insertion of the red fluorescent protein (RFP) in between the insertion site 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 alsT promoter is a promoter in B. subtilis repressed by TnrA which is active in the presence of low ammonium in the environment. TnrA will be deactivated in the presence of high ammonium in the environment. When TnrA is deactivated, alsT is no longer repressed. Ammonium is detected in the rotten meat and it can be used as a precursor of the rotting process and the increasing concentration of ammonium will trigger the TnrA - alsT reaction which means activating alsT promoter and activating the downstream genes.
A coding device to generate reporters (AmilCP, Lycopene, and AmilGFP) under regulation of sboA promoter. When 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.
Our sponsors:
