Part:BBa_K4813004

pFrmR-tdTomato formaldehyde sensing chromoprotein reporter

This composite part comprises several elements, including a formaldehyde sensing promoter (BBa_K2728001) called pFrmR, a strong ribosome binding site (BBa_B0034), an E. coli codon-optimized red chromoprotein protein tdTomato coding sequence (BBa_K4813001), and a strong double terminator (BBa_B0015). Additionally, the 5' and 3' ends of the composite part contain a 20-base pair overlap sequence designed for the NEBuilder HiFi assembly. This construct was cloned into the pUC19 vector at the EcoRI restriction site.

Fig. 1 Construct map showing the basic parts in this composite part.

The pFrmR promoter is a native formaldehyde-inducible promoter that is present in the genome of E. coli. It is a part of the formaldehyde detoxification mechanism in E. coli. It is regulated by the FrmR protein, which acts as a transcriptional repressor. In the presence of formaldehyde, the formaldehyde molecules bind to the FrmR proteins, causing their release from the repression of the pFrmR promoter. For our construct, we used the pFrmR part (BBa_K2728001), which was designed by Team iGEM18_BGIC-Global based on the original pFrmR part submitted by Team iGEM12_TMU-Tokyo (BBa_K749008).

Our project focuses on engineering E. coli bacteria to act as detectors of formaldehyde. These modified bacteria have the ability to change color when exposed to formaldehyde, providing a visible indication of its presence. This allows people to notice the presence of formaldehyde without requiring any special equipment.

We initially designed and ordered the construct as a gBlock from IDT. Unfortunately, due to time constraints, we were unable to proceed with the functional assay involving formaldehyde. This decision was due to our previous investigations, which showed that tdTomato gene does not exhibit a sufficiently deep color compared to dTomato gene. As a result, we prioritized testing the dTomato version of the detection device construct (BBa_K4813004).

To compare the chromoprotein coloration between dTomato and tdTomato, we conducted an experiment and documented the results. The detailed findings can be accessed here: (BBa_K4813000).

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1298
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1298
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1298
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1298
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 235