Difference between revisions of "Part:BBa K5152004"

Latest revision as of 02:56, 29 September 2024

PbrR-pPbr lead sensing chromoprotein reporter device

Our biosensor construct for detecting heavy metal lead is inspired by iGEM 2007 Team Brown (BBa_I721001). The regulatory protein PbrR controls the pPbr promoter. When lead is present, PbrR forms a dimer, activating the expression of downstream coding sequences.

We employed this construct in E. coli, which doesn't naturally express PbrR, so we co-expressed it. We used dTomato as the reporter gene, an RFP with chromoprotein properties, chosen for its visible colour to avoid the need for expensive equipment. This sequence, optimized by our 2023 team, ensures vibrant and stable expression. The illustration of the regulatory mechanism is shown below:

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

Usage and Biology

Lead Detection Functional Assay

Our biosensor effectively detected lead. After adding a final concentration of 100 µM lead (II) nitrate to the culture, cells exhibited visible red colouration in the pellet after 12 hours and significant culture colouration after 24 hours. We noted leaky expression with incubation times over 18 hours, suggesting a need to adjust the regulatory protein expression strength or incubation conditions.

Fig. 1: Biosensor cells exposed to 100 µM lead (II) nitrate showed an observable red colour in the pellets. While there is a noticeable difference, the red colouration in the culture form is less obvious.

Fig. 2: After 18 hours of incubation, the red colour in the pellet becomes visible. However, a slight red colour is observed in cells without added lead, indicating leaky expression.

Concentration Dependent Signals

We tested the biosensors with varying lead concentrations (0.01 µM to 1000 µM). After 24 hours, colour intensity was proportional to lead concentration, except at 500 µM and 1000 µM, likely due to toxicity affecting protein expression, as smaller pellet sizes were observed.

Fig. 3: The colour intensity of the biosensor cells increases with higher lead concentrations, suggesting that the biosensor design is concentration-dependent.

Reference

Borremans B, Hobman JL, Provoost A, Brown NL, van Der Lelie D. (2001) Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Bacteriol. 2001 Oct;183(19):5651-8. doi: 10.1128/JB.183.19.5651-5658.2001. PMID: 11544228; PMCID: PMC95457.

Wei W, Liu X, Sun P, Wang X, Zhu H, Hong M, Mao ZW, Zhao J. (2014) Simple whole-cell biodetection and bioremediation of heavy metals based on an engineered lead-specific operon. Environ Sci Technol. 2014 Mar 18;48(6):3363-71. doi: 10.1021/es4046567. Epub 2014 Mar 3. PMID: 24564581.

Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C. (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl. 2005 Apr 29;44(18):2715-2719. doi: 10.1002/anie.200462443. PMID: 15800869.

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
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 714

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K5152004 short</partinfo>
 Our biosensor construct for detecting heavy metal lead is inspired by iGEM 2007 Team Brown (<partinfo>BBa_I721001</partinfo>). The regulatory protein PbrR controls the <i>pPbr</i> promoter. When lead is present, PbrR forms a dimer, activating the expression of downstream coding sequences.
-We employed this construct in <i>E. coli</i>, which doesn't naturally express PbrR, so we co-expressed it. We used <i>dTomato</i> as the reporter gene, an RFP with chromoprotein properties, chosen for its visible colour to avoid the need for expensive equipment. This sequence, optimized by our 2023 team, ensures vibrant and stable expression.
+We employed this construct in <i>E. coli</i>, which doesn't naturally express PbrR, so we co-expressed it. We used <i>dTomato</i> as the reporter gene, an RFP with chromoprotein properties, chosen for its visible colour to avoid the need for expensive equipment. This sequence, optimized by our 2023 team, ensures vibrant and stable expression. The illustration of the regulatory mechanism is shown below:
+<html>
+<center>
+<img src="https://static.igem.wiki/teams/5152/part-registry/img-0687.png" width="600">
+</center>
+</html>
 Our project examined the expression profiles of several chromoproteins, including amilCP, cjBlue, tsPurple, eforRed, and dTomato. For more details, please refer to our wiki page.
@@ Line 24: / Line 30: @@
 <center>
 <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">
-<figcaption><u>Fig. 2: After 18 hours of incubation, the red colo山r in the pellet becomes visible. However, a slight red colo山r is observed in cells without added lead, indicating leaky expression.</u> </figcaption>
+<figcaption><u>Fig. 2: After 18 hours of incubation, the red colour in the pellet becomes visible. However, a slight red colour is observed in cells without added lead, indicating leaky expression.</u> </figcaption>
 </center>
 </html>
@@ Line 31: / Line 37: @@
 We tested the biosensors with varying lead concentrations (0.01 µM to 1000 µM).
-After 24 hours, colo山r intensity was proportional to lead concentration, except at 500 µM and 1000 µM, likely due to toxicity affecting protein expression, as smaller pellet sizes were observed.
+After 24 hours, colour intensity was proportional to lead concentration, except at 500 µM and 1000 µM, likely due to toxicity affecting protein expression, as smaller pellet sizes were observed.
 <html>
@@ Line 44: / Line 50: @@
 <b>Reference</b>
-Borremans B, Hobman JL, Provoost A, Brown NL, van Der Lelie D. Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Bacteriol. 2001 Oct;183(19):5651-8. doi: 10.1128/JB.183.19.5651-5658.2001. PMID: 11544228; PMCID: PMC95457.
+Borremans B, Hobman JL, Provoost A, Brown NL, van Der Lelie D. (2001) Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34.<i> J Bacteriol.</i>  2001 Oct;183(19):5651-8. doi: 10.1128/JB.183.19.5651-5658.2001. PMID: 11544228; PMCID: PMC95457.
-Wei, W., et al., Simple whole-cell biodetection and bioremediation of heavy metals based on an engineered lead-specific operon. Environ Sci Technol, 2014. 48(6): p. 3363-71.
+Wei W, Liu X, Sun P, Wang X, Zhu H, Hong M, Mao ZW, Zhao J. (2014) Simple whole-cell biodetection and bioremediation of heavy metals based on an engineered lead-specific operon. <i>Environ Sci Technol.</i> 2014 Mar 18;48(6):3363-71. doi: 10.1021/es4046567. Epub 2014 Mar 3. PMID: 24564581.
-Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C. An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl. 2005 Apr 29;44(18):2715-2719. doi: 10.1002/anie.200462443. PMID: 15800869.
+Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C. (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. <i>Angew Chem Int Ed Engl.</i> 2005 Apr 29;44(18):2715-2719. doi: 10.1002/anie.200462443. PMID: 15800869.