Part:BBa_K5152000
Constitutive tsPurple Chromoprotein Expression
Our team designed a 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 tsPurple coding sequence (BBa_K1033906), 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 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 colors 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 coloration and stable expression.
To simplify observation, we centrifuged 1 mL of the culture at 8000g for 2 minutes to form a pellet and then observed the color.
Successful expression was observed for most proteins after 12 hours of incubation, except cjBlue, which displayed color after 24 hours, making it less ideal for our project.
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.
From the result above, we found that at least 12 hours are needed for the cells to develop enough color 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 hardware to ensure accurate detection without extended incubation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 35
Illegal NheI site found at 58 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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