Part:BBa_K2936019
pBAD-lacZ alpha-pFrmR-AraC-CAT
The AraC protein would binds to AraBAD promoter, inhibiting its expression. At this time, only CAT gene is expressed in the pathway, then the culture added with TMB will turns blue. And when the pFrmR turn off, without repressor protein the AraBAD promoter activate, the LacZɑ expresses and the culture with ONPG would turns orange.Those parts form a bidirectional switch determined by formaldehyde concentration.
Short description
A bilateral switching element.
Description
We chosen the pFrmR, an engineered formaldehyde-inducible promoter, to expresses the genes when the concentration of CHOH in the environment reach a certain level. However, when testing the sensitive concentration of the FrmR promoter, we found that for the system cultured overnight, there was no obvious difference in the presence of with or without formaldehyde. That is to say, the promoter leakage was relatively serious. To control the genes to start or stop being expressed, we need a regulatory system. In consideration of the lactose operon has been used in light-dependent controlled lysis groups, Ara Operon is used in our pathways. The AraC protein would binds to AraBAD promoter, inhibiting its expression. At this time, only CAT gene is expressed in the pathway, then the culture added with TMB will turns blue. And when the pFrmR turn off, without repressor protein the AraBAD promoter activate, the LacZ alpha expresses and the culture with ONPG would turns orange.
Source
Escherichia coli and Parageobacillus.
Design consideration
The pBAD-LacZ alpha is the reverse complementary chain.
Future improvement
In order to solve the leak of pFrmR and achieve the ideal expression effect, our color reaction indicator system together with formaldehyde degradation group designed the amplification system to connect luxI to our group and put the gene expressing the enzyme of color reaction on PUC18.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 3178
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 3178
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 120
Illegal BamHI site found at 182
Illegal BamHI site found at 2421
Illegal XhoI site found at 3214 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 3178
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 3178
- 1000COMPATIBLE WITH RFC[1000]
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