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Revision as of 21:05, 25 September 2012

These are biobricks that our team submitted to the registry.

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 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 glucose, while the the double crossover mutants can. 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).

This plasmid backbone is designed to integrate a cloned insert into the Bacillus subtilis 168 chromosome at the sacA locus. The user inserts the fragment of interest between the prefix and suffix. 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 inability to produce red fluorescent protein for inserted plasmid. The red fluorescent protein cannot be produced in B.subtilis.

BBa_K818100 and BBa_K818200

These are the two most upregulated promoters detected from Bacillus subtilis 168 that are up regulated by the rotten meat's volatiles via microarray experiment. See the identification page for more information.


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 sboA promoter is activated, the downstream reporter will be produced.

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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