Part:BBa_K4813005

J23100 - dTomato red chromoprotein strong expression construct

This composite component comprises several elements, including a constitutive strong promoter (BBa_J23100), 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 feature a 20 base pair overlap sequence designed for the pUC19 EcoRI restriction site for NEBuilder HiFi assembly.

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

Our project aims to create a formaldehyde-sensing reporter. This composite part served three purposes:

1. Comparing expression levels of dTomato and tdTomato chromoproteins.

2. Providing a reference for expression levels in our formaldehyde-sensing reporter (BBa_K4813002).

3. Acting as a positive control for the assembly process. The colonies expressing this construct will be red in colour.

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 [1,2], 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, this one expressing the dTomato coding sequence(BBa_K4813005) and the other expressing tdTomato coding sequence (BBa_K4813006), and subsequently compared the colors of these two constructs as observed by the naked eye.

Fig. 2 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 this construct (Left) on the LB/Amp agar plates exhibited a deeper red color compared to those expressing tdTomato (Right) (Fig. 2). 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 the part expressing dTomato (BBa_K4813002) 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.

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/

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 27
  Illegal NheI site found at 50
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]