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Lac Operon Repressor
Codes for the production of the repressor protein that binds to the Lac Operator to prevent expression until induction of the media with IPTG. We have designed pUS270 to have the LacI under the pVeg promoter for strong constitutive expression in Bacillus.
pIP501 Rep
This gene codes for the Replicator protein that pairs with the Bacillus OriV and is essential to promote the independent replication of pUS270 in Bacillus.
E. coli OriV
Taken from pSB1C3, having this origin of replication will make it easier to construct the plasmid using E. coli as well as improve cloning efficiency downstream. Chosen because of the high copy number of pSB1C3.
Multiple Cloning Site
The multiple cloning site of pUS258 and other expression plasmids that we considered using either had too few restriction sites or sites that were incompatible for the efficient cloning with the BB Prefix and Suffix. So we have designed two highly diverse MCS’ that have 27 unique sites amongst them on either side of SF AmylCP-6 to promote simple cloning of parts from the Bio Brick Registry.
Antibiotic Resistance
Codes for Neomycin Phosphotransferase II that gives transformants resistance to both Neomycin and Kanamycin. Allows selective screening of transformants
Antibiotic Resistance
This region allows independent replication of the vector within Bacillus at a high copy number to provide more copies of the recombinant gene to maximise expression.
Lac Operator
Consists of the target sequence for the lac repressor protein to contribute to the IPTG induction system.
SuperFold AmilCP-6
SuperFold AmilCP-6 is a variant of amilCP which was used by the 2016 Sydney IGEM team and originally submitted to the registry by Team Uppsala Sweden in 2011. This variant has undergone site directed mutagenesis to increase the folding rate of the chromoprotein and enhance the blue colour. We are taking advantage of this protein to enable blue-white screening without the need for adding X-gal to the plates. Cloning with the two multiple cloning sites that flank SF AmilCP-6 essentially swaps it with the insert to leave the recombinant bacteria white rather than blue.
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