Device

Part:BBa_K4813022

Designed by: Lam Chung TIng   Group: iGEM23_HongKong-JSS   (2023-10-08)


Forward HxlR-dTomato formaldehyde sensing chromoprotein reporter

This composite component comprises several elements, including a modified RBS site in pET28a(+)-FGF2 plasmid (BBa_K4813019), a wild-type HxlR protein (BBa_K4813018), wild-type BRH1&2 binding sites for HxlR protein (BBa_K4813016), a wild-type promoter for Bacillus subtilis hxlAB operon (BBa_K4813017), a wild-type Bacillus subtilis RBS site (BBa_K4813021), an E. coli codon-optimized chromoprotein and red fluorescent protein dTomato coding sequence (BBa_K4813000), and a strong double terminator (B0015). Additionally, the 5' and 3' ends of the composite part feature a 20 base-pair overlap sequence designed for the pET28a(+)-FGF2 EcoRI and XbaI restriction sites for NEBuilder HiFi assembly (BBa_K4813010 & BBa_K4813011).

The HxlR protein is a formaldehyde-responsive transcription factor from Bacillus subtilis [1]. In the presence of formaldehyde, wild-type HxlR protein binds to two specific binding sites (BRH1&2) and activates the expression of the downstream hxlAB operon in Bacillus subtilis [1]. The expression of wild-type HxlR protein is naturally driven by a reverse promoter and we hardly found any previous research that has explored the possibility of having HxlR forwardly transcribed. In this composite component, to find out if a forwardly transcribed HxlR protein can still be expressed and retain its function, we specially designed to have HxlR protein driven by a forward T7 promoter. In our project, we utilise HxlR to promote the expression of a red-colored dTomato chromoprotein when formaldehyde is present. This composite component serves the purpose of detecting the presence of formaldehyde by inducing a color change in the host E. coli.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1374
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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