Difference between revisions of "Part:BBa K5152007"
| (3 intermediate revisions by the same user not shown) | |||
| Line 4: | Line 4: | ||
This part was used to create a cadmium-sensing biosensor for our project. | 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 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. |
| − | Our project goal is to develop biosensors for heavy metals without needing special techniques or expensive equipment. We chose chromoproteins for their visible | + | 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. | Our project also examined several chromoproteins, including amilCP, cjBlue, tsPurple, eforRed, and dTomato. For more details, please refer to our wiki page. | ||
| Line 13: | Line 13: | ||
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. | 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 | + | 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. |
<html> | <html> | ||
<center> | <center> | ||
| − | <img src="https://static.igem.wiki/teams/5152/part-registry/18-pyoda-functional-leaky-expression.webp" alt="200 uM cadmium pYodA" width=" | + | <img src="https://static.igem.wiki/teams/5152/part-registry/18-pyoda-functional-leaky-expression.webp" alt="200 uM cadmium pYodA" width="400"> |
| − | <figcaption><u>Fig. 1: The pellets showed similar blue | + | <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> |
</center> | </center> | ||
</html> | </html> | ||
| + | |||
| + | |||
| + | <b>Reference</b> | ||
| + | |||
| + | [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. | ||
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]