Part:BBa_K4813001

tdTomato - red fluorescent protein codon optimized for E. coli

The coding sequence provided corresponds to the protein tdTomato, which is responsible for generating red fluorescence. This engineered protein is formed by fusing two dTomato proteins. tdTomato is widely used in various imaging applications due to its bright red fluorescence. [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 tdTomato 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_K4813004), while the other part (BBa_K4813006) acted as a positive control, where the expression of tdTomato was driven by a strong constitutive promoter (BBa_J23100).

In our project, we specifically emphasized the chromoprotein property of the protein rather than its fluorescence. Despite literature suggesting that tdTomato exhibits a brighter expression, our findings demonstrated that dTomato displayed a higher intensity of red color in bacterial colonies. This led us to select dTomato as the preferred reporter for our subsequent project steps, which involve detecting formaldehyde. The decision was driven by the importance of enabling naked-eye observation of the results by non-experts, eliminating the requirement for fluorescence equipment.

Reference

[1] 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 [1], 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 dTomato BBa_K4813000 for our functional assays instead of this part.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1066
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1066
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1066
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1066
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3