From 2012.igem.org

Latest revision as of 18:35, 26 October 2012

These are the biobricks that team Groningen submitted to the registry.

The pink heart means 'group favorite' and 'W' means works.

BBa_K818000
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 E. coli. In Bacillus subtilis, the plasmid integrates at sacA. 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.

BBa_K818100 and BBa_K818200
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.

BBa_K818300
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.

BBa_K818400, BBa_K818500, and BBa_K818600
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.

BBa_K818210 and BBa_K818310 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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