Difference between revisions of "Part:BBa K4813000"
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This part encodes a protein called dTomato, which is a red fluorescence protein. It forms a dimer and has a molecular weight of around 27.0 kDa. This protein originates from an anemone species called ''Discosoma'' [1].
 
This part encodes a protein called dTomato, which is a red fluorescence protein. It forms a dimer and has a molecular weight of around 27.0 kDa. This protein originates from an anemone species called ''Discosoma'' [1].
  
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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 dTomato 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 (<partinfo>BBa_K4813003</partinfo>), while the other part (<partinfo>BBa_K4813005</partinfo>) acted as a positive control, where the expression of ''dTomato'' was driven by a strong constitutive promoter (<partinfo>BBa_J23100</partinfo>).  
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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 dTomato 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 (<partinfo>BBa_K4813002</partinfo>), while the other part (<partinfo>BBa_K4813005</partinfo>) acted as a positive control, where the expression of ''dTomato'' was driven by a strong constitutive promoter (<partinfo>BBa_J23100</partinfo>).  
  
 
<b>Reference</b>
 
<b>Reference</b>
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Although the fluorescence protein database suggests that tdTomato has better fluorescence emission compared to dTomato [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.
 
Although the fluorescence protein database suggests that tdTomato has better fluorescence emission compared to dTomato [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.
  
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To address this, we have generated two composite parts, one expressing the ''dTomato'' coding sequence(<partinfo>BBa_K4813002</partinfo>) and the other expressing ''tdTomato'' coding sequence (<partinfo>BBa_K4813004</partinfo>), and subsequently compared their colors as observed by the naked eye.
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To address this, we have generated two composite parts, one expressing the ''dTomato'' coding sequence(<partinfo>BBa_K4813005</partinfo>) and the other expressing ''tdTomato'' coding sequence (<partinfo>BBa_K4813006</partinfo>), and subsequently compared their colors as observed by the naked eye.
  
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[[Image:logo-png.jpg]]
 
  
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The bacterial colonies expressing dTomato on the LB/Amp agar plates (Fig. 1) exhibited a deeper red color compared to those expressing tdTomato (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.
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<html><center>
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<img src="https://static.igem.wiki/teams/4813/wiki/experiments/plate-expressing-j23100-dtom.jpeg" alt="The plate with colonies expressing dTomato" width="300">
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<img src="https://static.igem.wiki/teams/4813/wiki/experiments/plate-expressing-j23100-tdtom.jpeg" alt="The plate with colonies expressing tdTomato" width="300">
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<figcaption><u>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.</u> </figcaption>
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</center>
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</html>
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 +
 
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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 this part 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.
 
Consequently, we chose this part 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.
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<partinfo>BBa_K4813000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4813000 SequenceAndFeatures</partinfo>
  
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<!-- Add more about the biology of this part here
 
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<!-- -->
 
  
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==Contribution==
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*'''Group:'''  iGEM24_HongKong-JSS
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===Expression characterization===
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===Lead biosensor===
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Our project this year focuses on creating biosensors for heavy metals. We utilized dTomato in our lead biosensor designs (<partinfo>BBa_K5152004</partinfo>).
 +
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Our results show that dTomato expression is distinct and easily visible. The color intensity changes noticeably with varying concentrations. In our lead biosensor, the culture's color intensity increases with higher lead concentrations, enhancing the biosensor's applicability.
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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. 1: The dTomato color is easily distinguishable from the culture solution.</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/pb-1-100-um.webp" alt="Pb concentration" width="500">
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<figcaption><u>Fig. 2: The color 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>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 03:35, 29 September 2024


dTomato fluoresence protein Codon Optimized for E coli

This part encodes a protein called dTomato, which is a red fluorescence protein. It forms a dimer and has a molecular weight of around 27.0 kDa. This protein originates from an anemone species called Discosoma [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 dTomato 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_K4813002), while the other part (BBa_K4813005) acted as a positive control, where the expression of dTomato was driven by a strong constitutive promoter (BBa_J23100).

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/


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 [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, 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.


The plate with colonies expressing dTomato The plate with colonies expressing tdTomato
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 this part 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution

  • Group: iGEM24_HongKong-JSS

Expression characterization

Lead biosensor

Our project this year focuses on creating biosensors for heavy metals. We utilized dTomato in our lead biosensor designs (BBa_K5152004).

Our results show that dTomato expression is distinct and easily visible. The color intensity changes noticeably with varying concentrations. In our lead biosensor, the culture's color intensity increases with higher lead concentrations, enhancing the biosensor's applicability.

100 uM Pb 18 hours
Fig. 1: The dTomato color is easily distinguishable from the culture solution.

Pb concentration
Fig. 2: The color intensity of the biosensor cells increases with higher lead concentrations, suggesting that the biosensor design is concentration-dependent.