Difference between revisions of "Part:BBa K5152009"

Latest revision as of 00:08, 27 September 2024

pZnt zinc sensing chromoprotein reporter device

We designed this part to create our zinc biosensor, using a zinc-inducible promoter, pZnt, native to E. coli and regulated by the ZntR activator protein. Inspired by iGEM 2009 Team Groningen, we didn't co-express ZntR since it's already present in E. coli.

The construct includes eforRed, a chromoprotein, as the reporter signal. Our goal is to develop a user-friendly and cost-effective biosensor, so the visible red colour acts as a signal without needing expensive equipment or special techniques.

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

Usage and Biology

Zinc Detection Functional Assay

We successfully cloned the construct and validated it using colony PCR. However, most colonies turned red without zinc due to leaky expression. We proceeded by selecting white colonies and adding 200 µM zinc (II) chloride.

After 12 hours of incubation, both the negative control and the zinc setup showed red colouration in the pellets. Although the red was slightly deeper with zinc, the difference wasn't significant, so we couldn't confirm the biosensor's function. Further investigation is needed to refine this design.

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

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]

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 __NOTOC__
 <partinfo>BBa_K5152009 short</partinfo>
-This device contains the zinc-inducible promoter pZnt, natively found in E. coli, to drive the expression of the chromoprotein eforRed 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 zinc, indicating that the promoter's repression needs to be strengthened in future designs to improve specificity and reduce background expression.
+We designed this part to create our zinc biosensor, using a zinc-inducible promoter, <i>pZnt</i>, native to <i>E. coli</i> and regulated by the ZntR activator protein. Inspired by iGEM 2009 Team Groningen, we didn't co-express ZntR since it's already present in <i>E. coli</i>.
+The construct includes eforRed, a chromoprotein, as the reporter signal. Our goal is to develop a user-friendly and cost-effective biosensor, so the visible red colour acts as a signal without needing expensive equipment or special techniques.
+Our project also examined chromoproteins like amilCP, cjBlue, tsPurple, eforRed, and dTomato. For more details, please refer to our wiki page.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
-<!-- -->
+<b>Zinc Detection Functional Assay</b>
-<span class='h3bb'>Sequence and Features</span>
+We successfully cloned the construct and validated it using colony PCR. However, most colonies turned red without zinc due to leaky expression. We proceeded by selecting white colonies and adding 200 µM zinc (II) chloride.
+After 12 hours of incubation, both the negative control and the zinc setup showed red colouration in the pellets. Although the red was slightly deeper with zinc, the difference wasn't significant, so we couldn't confirm the biosensor's function. Further investigation is needed to refine this design.
+<html>
+<center>
+<img src="https://static.igem.wiki/teams/5152/part-registry/19-pznt-functional-leaky-expression.webp" alt="200 uM zinc pZnt" width="400">
+<figcaption><u>Fig. 1: The pellets showed similar blue colouration both with and without zinc, so the biosensor's function couldn't be confirmed.</u> </figcaption>
+</center>
+</html>
+<span class='h3bb'><b>Sequence and Features</b></span>
 <partinfo>BBa_K5152009 SequenceAndFeatures</partinfo>