Difference between revisions of "Part:BBa K5152007"

 
 
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<partinfo>BBa_K5152007 short</partinfo>
 
<partinfo>BBa_K5152007 short</partinfo>
  
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This device uses pYodA, a cadmium-inducible promoter natively found in E. coli, to drive the expression of the chromoprotein amilCP as the reporter signal. As a result, we did not co-express any regulatory proteins in our design. However, our results showed significant leaky expression even in the absence of cadmium, suggesting that the promoter's repression needs to be strengthened in future designs to improve specificity and reduce background expression.
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This part was used to create a cadmium-sensing biosensor for our project.
  
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Our literature review identified pYodA as a cadmium-inducing promoter regulated by the yodA protein in <i>E. coli</i> (A. Puškarova et al., 2002). We noted that iGEM 2012 Team NYMU-Taipei previously used this promoter, but there is limited documentation in the part registry. We aim to test pYodA in our design.
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Our project goal is to develop biosensors for heavy metals without needing special techniques or expensive equipment. We chose chromoproteins for their visible colours. This biosensor uses the amilCP chromoprotein, which turns cells blue in the presence of cadmium.
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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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We successfully cloned the construct and validated it using colony PCR, but in the transformants, most colonies turned blue without cadmium due to leaky expression. We proceeded with functional studies by selecting colonies that remained white and adding 200 µM cadmium chloride.
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After 12 hours of incubation, both the negative control and the setup with cadmium showed blue colouration in the harvested pellets. Although the blue was consistently more intense with cadmium, the difference wasn't significant. This suggests that our pYodA construct has potential as a cadmium biosensor, but further investigation and modification are needed.
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<html>
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<center>
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<img src="https://static.igem.wiki/teams/5152/part-registry/18-pyoda-functional-leaky-expression.webp" alt="200 uM cadmium pYodA" width="400">
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<figcaption><u>Fig. 1: The pellets showed similar blue colouration both with and without cadmium, so the biosensor's function couldn't be confirmed.</u> </figcaption>
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<b>Reference</b>
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[1] Puškarova A, Ferianc P, Kormanec J, Homerova D, Farewell A, Nyström T. Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli. Microbiology (Reading). 2002 Dec;148(Pt 12):3801-3811. doi: 10.1099/00221287-148-12-3801. PMID: 12480884.
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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_K5152007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5152007 SequenceAndFeatures</partinfo>
  

Latest revision as of 00:11, 27 September 2024

pYodA cadmium sensing chromoprotein reporter device

This part was used to create a cadmium-sensing biosensor for our project.

Our literature review identified pYodA as a cadmium-inducing promoter regulated by the yodA protein in E. coli (A. Puškarova et al., 2002). We noted that iGEM 2012 Team NYMU-Taipei previously used this promoter, but there is limited documentation in the part registry. We aim to test pYodA in our design.

Our project goal is to develop biosensors for heavy metals without needing special techniques or expensive equipment. We chose chromoproteins for their visible colours. This biosensor uses the amilCP chromoprotein, which turns cells blue in the presence of cadmium.

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

We successfully cloned the construct and validated it using colony PCR, but in the transformants, most colonies turned blue without cadmium due to leaky expression. We proceeded with functional studies by selecting colonies that remained white and adding 200 µM cadmium chloride.

After 12 hours of incubation, both the negative control and the setup with cadmium showed blue colouration in the harvested pellets. Although the blue was consistently more intense with cadmium, the difference wasn't significant. This suggests that our pYodA construct has potential as a cadmium biosensor, but further investigation and modification are needed.

200 uM cadmium pYodA
Fig. 1: The pellets showed similar blue colouration both with and without cadmium, so the biosensor's function couldn't be confirmed.


Reference

[1] Puškarova A, Ferianc P, Kormanec J, Homerova D, Farewell A, Nyström T. Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli. Microbiology (Reading). 2002 Dec;148(Pt 12):3801-3811. doi: 10.1099/00221287-148-12-3801. PMID: 12480884.


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]