Part:BBa_K5152003

Constitutive eforRed Chromoprotein Expression

Our team designed this composite part to verify and compare the expression of chromoprotein reporters. Our project aims to develop biosensors for heavy metals that don't require specialized equipment for measurement. Therefore, we chose chromoproteins instead of fluorescence or luciferase.

This composite part includes: A constitutive strong promoter (BBa_J23100), a strong ribosome binding site (BBa_B0034), the eforRed coding sequence (BBa_K592012), and a strong double terminator (BBa_B0015). Additionally, the 5' and 3' ends feature a 20 base pair overlap sequence for NEBuilder HiFi assembly using the pUC19 PstI and EcoRI restriction sites. The graphical illustration of this construct design is shown below:

The purpose of this part in our project includes:

1. Ensuring successful protein expression in our E. coli strain under lab conditions.
2. Verifying that the expressed protein colours match expected results.
3. Using these constructs to validate our cloning process and experimental setup.
4. Employing these constructs as positive controls for future experiments.

Usage and Biology

Successful Expression of Chromoproteins

We selected five chromoproteins as potential biosensor candidates: tsPurple (BBa_K1033906), eforRed (BBa_K592012), cjBlue (BBa_K592011), amilCP (BBa_K592009), and dTomato (BBa_K4813000). Our 2023 team constructed dTomato, which showed promising colouration and stable expression. The expression constructs of the above chromoproteins are tsPurple (BBa_K5152000), eforRed (BBa_K5152003), cjBlue (BBa_K5152001), amilCP (BBa_K5152002), and dTomato (BBa_K4813005).

To simplify observation, we centrifuged 1 mL of the culture at 8000g for 2 minutes to form a pellet and then observed the colour.

Fig. 1 The pellets we obtained from our E. coli cells expressing chromoproteins.

Successful expression was observed for most proteins after 12 hours of incubation, except cjBlue, which displayed colour after 24 hours, making it less ideal for our project.

Fig. 2 After 12 hours of incubation, most of the cjBlue expressing cells did not display the expected colour.

Fig. 3 After incubating for at least 24 hours, the cjBlue plates have finally displayed the expected colouration.

Expression Properties

We characterized the expression properties by focusing on the time required for visible results.

Cells were cultured and harvested at 4, 8, 12, 18, and 24 hours. At each time point, 1 mL of culture was centrifuged at 8,000 g for 2 minutes to obtain cell pellets for observation.

Diagrams below show results from the 12-hour and 18-hour time points for reference.

Fig. 4 Pellets of E. coli cells expressing chromoproteins after 12 and 18 hours of incubation at 37°C, 180 rpm.

From the result above, we found that at least 12 hours are needed for the cells to develop enough colour for clear reading. While waiting around 18 hours gives more noticeable results, we noticed that in cells with metal-sensing constructs, longer incubation can cause unwanted chromoprotein expression, leading to false positives. It's important to set a specific time and threshold for our measurement to ensure accurate detection without extended incubation.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 35
  Illegal NheI site found at 58
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]