Difference between revisions of "Part:BBa K5152006"

 
 
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<partinfo>BBa_K5152006 short</partinfo>
 
<partinfo>BBa_K5152006 short</partinfo>
  
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This device includes the cadmium-inducible pCadA promoter and co-expresses its repressor MerR, which has been specifically modified to bind cadmium. In the presence of cadmium, the pCadA promoter activates the expression of the downstream chromoprotein amilCP, serving as the reporter signal. This device is specifically designed to detect the presence of cadmium in the environment.
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This part was used to create a cadmium-sensing biosensor for our project, inspired by iGEM 2015 Team SCUT (<partinfo>BBa_K1724000</partinfo>).
  
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The <i>pCadA</i> promoter is cadmium-activated and normally repressed by MerR, which binds mercury. In our design, we used a modified MerR (<partinfo>BBa_K1724001</partinfo>) that binds to cadmium, making the system sensitive to cadmium. Interestingly, although MerR in its native form is an activator for the mercury-sensitive pMerT promoter, the SCUT Team's report indicates that the modified MerR acts as a repressor for the pCadA promoter [1]. We co-expressed the modified MerR under a medium-strength promoter to enhance sensitivity. The cell strain we used was TOP10 <i>E. coli</i>.
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Since the goal of our project is to develop biosensors for heavy metals that don't require special techniques or expensive equipment, we used chromoproteins for their visible colours. This biosensor uses the amilCP chromoprotein, which turns cells blue in the presence of cadmium.
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The illustration of the regulatory mechanism of our design is shown below:
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<center>
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<img src="https://static.igem.wiki/teams/5152/part-registry/img-0691.png" width="600">
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</html>
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Our project also examined several chromoproteins, including amilCP, cjBlue, tsPurple, eforRed, and dTomato. For more details, please refer to our wiki page.
  
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===Usage and Biology===
 
===Usage and Biology===
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<b>Cadmium Detection Functional Assay</b>
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Our biosensor design effectively detected cadmium. After adding 200 µM cadmium (II) chloride to the culture, the cells showed visible blue colouration in the pellet after 12 hours. However, we consistently observed that extending the incubation time caused the negative control to also turn blue, indicating leaky expression. Therefore, it's important to carefully choose specific timing and conditions to avoid false positives.
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<img src="https://static.igem.wiki/teams/5152/part-registry/17ab-merr-pcada-functional.webp" alt="100 uM Pb 12 hours" width="600">
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<figcaption><u>Fig. 1: Biosensor cells exposed to 200 µM cadmium (II) chloride displayed an observable blue colour in the pellets. While there's a noticeable difference, the blue colouration in the culture is less distinct.</u> </figcaption>
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<b>Reference</b>
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[1] Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. <i>FEMS microbiology reviews</i>, 27(2-3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2
  
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<span class='h3bb'><b>Sequence and Features</b></span>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5152006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5152006 SequenceAndFeatures</partinfo>
  

Latest revision as of 03:39, 29 September 2024

MerR-pCadA cadmium sensing chromoprotein reporter device

This part was used to create a cadmium-sensing biosensor for our project, inspired by iGEM 2015 Team SCUT (BBa_K1724000).

The pCadA promoter is cadmium-activated and normally repressed by MerR, which binds mercury. In our design, we used a modified MerR (BBa_K1724001) that binds to cadmium, making the system sensitive to cadmium. Interestingly, although MerR in its native form is an activator for the mercury-sensitive pMerT promoter, the SCUT Team's report indicates that the modified MerR acts as a repressor for the pCadA promoter [1]. We co-expressed the modified MerR under a medium-strength promoter to enhance sensitivity. The cell strain we used was TOP10 E. coli.

Since the goal of our project is to develop biosensors for heavy metals that don't require special techniques or expensive equipment, we used chromoproteins for their visible colours. This biosensor uses the amilCP chromoprotein, which turns cells blue in the presence of cadmium.

The illustration of the regulatory mechanism of our design is shown below:

Our project also examined several chromoproteins, including amilCP, cjBlue, tsPurple, eforRed, and dTomato. For more details, please refer to our wiki page.

Usage and Biology

Cadmium Detection Functional Assay

Our biosensor design effectively detected cadmium. After adding 200 µM cadmium (II) chloride to the culture, the cells showed visible blue colouration in the pellet after 12 hours. However, we consistently observed that extending the incubation time caused the negative control to also turn blue, indicating leaky expression. Therefore, it's important to carefully choose specific timing and conditions to avoid false positives.

100 uM Pb 12 hours
Fig. 1: Biosensor cells exposed to 200 µM cadmium (II) chloride displayed an observable blue colour in the pellets. While there's a noticeable difference, the blue colouration in the culture is less distinct.

Reference

[1] Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS microbiology reviews, 27(2-3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 350
    Illegal AgeI site found at 452
  • 1000
    COMPATIBLE WITH RFC[1000]