Part:BBa_K4813002

pFrmR-dTomato formaldehyde sensing chromoprotein reporter device

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 dTomato coding sequence (BBa_K4813000), 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.

Usage and Biology

The E. coli expressing this construct was able to show colour change with the presence of formaldehyde

Validation of clones

We obtained the part constructs in the form of gBlocks from IDT. We then performed HiFi assembly (NEB) to assemble these constructs in pUC19 vectors. To verify the clones, we ordered pUC19 M13 PCR primers and performed colony PCR on the resulting transformants. Initially, we faced several unsuccessful attempts at cloning. However, after multiple trials, we successfully cloned the desired construct and transformed it into E. coli.

Fig. 2 The gel result of colony PCR for verifying the clones of this part. Lane 1 is the DNA ladder (1kbp+ NEB), Lane 2-8 are the colonies, showing the expected band size of ~1.1kbp. Lane 9 is the no template control.

Figure 2 shows the gel electrophoresis result of a successful cloning attempt from colony PCR, showing the presence of a band of the predicted size (~1.1kbp) corresponding to the M13 amplification of our inserted constructs. It is worth mentioning that during our observations, we noticed that some colonies on the plate showed a pink colour, indicating potential issues with leaky expression. Therefore, we decided to select a white-colored colony for further investigation and proceed with our functional assay involving formaldehyde.

Functional assay with formaldehyde

The E. coli strain expressing the construct was then cultured in 10 mL of LB broth with ampicillin for a duration of 12 hours. During this period, formaldehyde was added in concentrations of 500 uM and 1000 uM. Following the incubation, the culture was harvested and subjected to centrifugation, resulting in the formation of a pellet for easier observation of any color changes.

Fig. 3 The left pellet contains E. coli expressing BBa_K4813002 construct without any formaldehyde treatment, maintaining a white color. Conversely, the right pellet shows E. coli expressing the same construct but treated with 500 uM formaldehyde, resulting in a pink coloration.

Figure 3 shows the results obtained from the control sample with no formaldehyde (Left) and the sample with 500 uM formaldehyde (Right). In the 500 uM formaldehyde setup, a noticeable pink coloration is observed in the pellet, whereas the control sample without formaldehyde retains its white colour. This result confirms that our construct can show a color change in our engineered E. coli strain in the presence of formaldehyde.

Furthermore, we observed that the culture with the 1000 uM formaldehyde setup showed no turbidity. This suggests that the concentration of 1000 uM may have inhibited the growth of the E. coli strain.

In future work, we aim to study how the color intensity of our constructs varies with different concentrations of formaldehyde. We also plan to enhance the sensitivity of our device to detect even lower levels of formaldehyde.

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]