Part:BBa_K4813024

Forward HxlR-K13A-dTomato formaldehyde sensing chromoprotein reporter

This composite component comprises several elements, including a modified RBS site in pET28a(+)-FGF2 plasmid (BBa_K4813019), a HxlR protein with a K13A mutation (BBa_K4813020), 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 (BBa_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]. According to literature, when the 13th amino acid in HxlR protein, Lysine, was replaced by Alanine, mutated HxlR-K13A protein showed a more optimal conformation for DNA binding and transcription activation than the wild-type HxlR protein [1].

In this composite component, we incorporated the mutated K13A version of HxlR gene to examine if HxlR-K13A can lead to an enhanced expression of a red-colored dTomato chromoprotein when formaldehyde is present. In our project, this composite component serves the purpose of detecting the presence of formaldehyde by inducing a color change in the host E. coli. In another composite component developed by us (BBa_K), we made the naturally backward transcribed wild-type HxlR protein to become forwardly transcribed driven by a forward T7 promoter. Hence, the mutated HxlR-K13A in this composite component is also forwardly transcribed driven by the same forward T7 promoter as a comparison.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]