Part:BBa_K4813000

dTomato fluoresence protein Codon Optimized for E coli

This part encodes a protein called dTomato, which is a red fluorescence protein. It forms a dimer and has a molecular weight of around 27.0 kDa. This protein originates from an anemone species called Discosoma [1].

To enhance its performance for our project, we used IDT codon optimization tools to optimize the sequence. This part is the optimized version of the native dTomato sequence obtained from NCBI. We incorporated this optimized sequence into two composite parts. One part served as a reporter gene for detecting formaldehyde with pFrmR formaldehyde sensing promoter (BBa_K4813002), while the other part (BBa_K4813005) acted as a positive control, where the expression of dTomato was driven by a strong constitutive promoter (BBa_J23100).

Reference

[1] dTomato at fpbase. FPbase. Accessed 20 June 2023 https://www.fpbase.org/protein/dtomato/

[2] tdTomato at fpbase. FPbase. Accessed 20 June 2023 https://www.fpbase.org/protein/tdtomato/

Usage and Biology

Comparing the colouration of optimized dTomato BBa_K4813000 and tdTomato BBa_K4813001

Although the fluorescence protein database suggests that tdTomato has better fluorescence emission compared to dTomato [2], it does not provide information about their chromoprotein properties. Since our project aims to create a user-friendly device for monitoring formaldehyde levels without requiring specialized equipment, we are looking for a chromoprotein that produces a more visible color to the naked eye.

To address this, we have generated two composite parts, one expressing the dTomato coding sequence(BBa_K4813005) and the other expressing tdTomato coding sequence (BBa_K4813006), and subsequently compared their colors as observed by the naked eye.

Fig. 1 After 12 hours incubation, the plate (left) with colonies expressing dTomato RFP is showing a significantly higher intensity of red colour than the plate (right) with colonies expressing tdTomato.

The bacterial colonies expressing dTomato on the LB/Amp agar plates (left) exhibited a deeper red color compared to those expressing tdTomato (right) (Fig. 1). We hypothesized that it is due to the larger size of tdTomato (which is composed of two dTomato proteins) may require more energy for expression by the E. coli cells. As a result, this increased energy demand potentially leads to a slower growth rate of the cells expressing tdTomato and a lower overall protein expression level within the colonies. To confirm our hypothesis, further investigation will be necessary.

Consequently, we chose this part for the functional assay, as it proved to be more obvious in detecting the change of colour in E. coli with the presence 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]

Contribution

• Group: iGEM24_HongKong-JSS

Lead biosensor

Our project this year focuses on creating biosensors for heavy metals. We utilized dTomato in our lead biosensor designs (BBa_K5152004).

Our results show that dTomato expression is distinct and easily visible. The color intensity changes noticeably with varying concentrations. In our lead biosensor, the culture's color intensity increases with higher lead concentrations, enhancing the biosensor's applicability.

Fig. 1: The dTomato color is easily distinguishable from the culture solution.

Fig. 2: The color intensity of the biosensor cells increases with higher lead concentrations, suggesting that the biosensor design is concentration-dependent.