Difference between revisions of "Part:BBa K4813005"
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[2] tdTomato at fpbase. FPbase. Accessed 20 June 2023 https://www.fpbase.org/protein/tdtomato/  
 
[2] tdTomato at fpbase. FPbase. Accessed 20 June 2023 https://www.fpbase.org/protein/tdtomato/  
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==Contribution==
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*'''Group:'''  iGEM24_HongKong-JSS
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===Lead biosensor===
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Our team used this part to construct a lead biosensor with <i>E. coli</i>. We aimed to create biosensors visible to the naked eye without special equipment, so we employed the dTomato chromoprotein, previously optimized by our team, as the reporter gene (<partinfo>BBa_K5152004</partinfo>).
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We successfully validated our biosensor's ability to detect lead (100 µM lead (II) nitrate). The medium turns red due to dTomato expression. We obtained pellets from 1 mL of biosensor cultures after spinning at 8,000 g for 2 minutes.
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<html>
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<center>
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<img src="https://static.igem.wiki/teams/5152/part-registry/13-ab-ppbr-functional-100-um.webp" alt="100 uM Pb 12 hours" width="600">
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<figcaption><u>Fig. 1: Biosensor cells exposed to 100 µM lead (II) nitrate showed an observable red colour in the pellets.
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While there is a noticeable difference, the red colouration in the culture form is less obvious.</u> </figcaption>
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</center>
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</html>
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<html>
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<center>
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<img src="https://static.igem.wiki/teams/5152/part-registry/14-ppbr-100-um-after-18-hours.webp" alt="100 uM Pb 18 hours" width="600">
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<figcaption><u>Fig. 2: After 18 hours of incubation, the red colour in the pellet becomes visible. However, a slight red colour is observed in cells without added lead, indicating leaky expression.</u> </figcaption>
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</center>
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</html>
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The colour signal in the pellet becomes distinguishable after 12 hours of incubation, while the culture's signal is significant after 18 hours with lead. However, leaky red expression is observed if incubation exceeds 18 hours.
 +
 +
We also demonstrated that the reporter is concentration-dependent; the color intensity increases with higher lead concentrations. The figure below shows our biosensor after incubating with 0, 0.01, 0.1, 1, and 10 µM lead (II) nitrate solutions for 24 hours. The color differences are easily distinguishable.
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<html>
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<center>
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<img src="https://static.igem.wiki/teams/5152/part-registry/pb-concentration-0-10.webp" alt="0-10uM Pb 24 hrs" width="600">
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<figcaption><u>Fig. 3: The colour intensity of the biosensor cells increases with higher lead concentrations,
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suggesting that the biosensor design is concentration-dependent.</u> </figcaption>
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</center>
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</html>
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This suggests that our biosensor design is effective and could have broad applications.
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 12:19, 28 September 2024


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.


Construct map of K4813005
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.


The plate with colonies expressing dTomato The plate with colonies expressing tdTomato
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/

Contribution

  • Group: iGEM24_HongKong-JSS

Lead biosensor

Our team used this part to construct a lead biosensor with E. coli. We aimed to create biosensors visible to the naked eye without special equipment, so we employed the dTomato chromoprotein, previously optimized by our team, as the reporter gene (BBa_K5152004).

We successfully validated our biosensor's ability to detect lead (100 µM lead (II) nitrate). The medium turns red due to dTomato expression. We obtained pellets from 1 mL of biosensor cultures after spinning at 8,000 g for 2 minutes.

100 uM Pb 12 hours
Fig. 1: Biosensor cells exposed to 100 µM lead (II) nitrate showed an observable red colour in the pellets. While there is a noticeable difference, the red colouration in the culture form is less obvious.

100 uM Pb 18 hours
Fig. 2: After 18 hours of incubation, the red colour in the pellet becomes visible. However, a slight red colour is observed in cells without added lead, indicating leaky expression.

The colour signal in the pellet becomes distinguishable after 12 hours of incubation, while the culture's signal is significant after 18 hours with lead. However, leaky red expression is observed if incubation exceeds 18 hours.

We also demonstrated that the reporter is concentration-dependent; the color intensity increases with higher lead concentrations. The figure below shows our biosensor after incubating with 0, 0.01, 0.1, 1, and 10 µM lead (II) nitrate solutions for 24 hours. The color differences are easily distinguishable.

0-10uM Pb 24 hrs
Fig. 3: The colour intensity of the biosensor cells increases with higher lead concentrations, suggesting that the biosensor design is concentration-dependent.

This suggests that our biosensor design is effective and could have broad applications.

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]