Difference between revisions of "Part:BBa K2447000"

Latest revision as of 19:40, 17 December 2021

Extracellular phosphate sensor with GFP reporter

PhoB and PhoR proteins are part of the Pho regulon in E. coli. When a low concentration of extracellular phosphate ions is present, PhoR can phosphorylate PhoB to form an active phosphorylated-PhoB. PhoB promoter is activated as a result of active binding of phosphorylated-PhoB, resulting in downstream expression of GFP. When a high concentration of extracellular phosphate ions is present, PhoR will dephosphorylate PhoB, and therefore inactivating it, and repressing the PhoB promoter for GFP expression.

Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.

Improvement over previous iGEM part BBa_K116404 (NYMU Taipei 2008)

The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbone and subsequently characterized in E. coli MG 1655. These cells were grown in LB medium before resuspension in MOPS medium (a minimal nutrient medium) for characterization with a micro-plate reader. Varying concentrations of phosphate ions from 0 to 1000 uM were added and GFP expression was monitored.

By replacing the weaker RBS BBa_B0032 of the original part with a stronger RBS BBa_B0034, we have successfully constructed an improved phosphate sensor-GFP reporter. Our part shows, on average, a 40-fold increase in GFP expression (Figure 3) when compared to the previous version of the construct (http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate). The original phosphate construct is also insensitive to high phosphate concentrations above 50 µM where similar levels of GFP expression are observed (Figure 4). Unlike the previous construct, our improved phosphate construct is much more sensitive to various phosphate concentrations from 0 to 1000 µM, particularly at phosphate concentrations above 50 µM.

Figure 2: Side by side comparison of our improved construct Bba_K2447000 & the original construct BBa_K116404. Our proposed part exhibited greater GFP expression at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50 uM.

Figure 3: Our improved phosphate construct is much more sensitive to phosphate concentrations above 50 uM, unlike the original construct.

Figure 4: GFP expression by the original phosphate construct designed by the Taiwanese team.

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
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1162

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 <partinfo>BBa_K2447000 short</partinfo>
-PhoB and PhoR proteins are part of the Pho regulon inherent in E.coli. When low concentration of extracellular phosphate ions is present, PhoR can phosphorylate PhoB to form active phosphorylated-PhoB. PhoB promoter is activated as a result of active binding of phosphorylated-PhoB, resulting in downstream expression of GFP. When high concentration of extracellular phosphate ions is present, PhoR will dephosphorylate phosphorylated-PhoB, and therefore inactivating it, and repressing PhoB promoter for downstream expression of GFP. [[Image:Phosphate1.png|thumb|center|500px|PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP]]
+PhoB and PhoR proteins are part of the Pho regulon in <i>E. coli</i>. When a low concentration of extracellular phosphate ions is present, PhoR can phosphorylate PhoB to form an active phosphorylated-PhoB. PhoB promoter is activated as a result of active binding of phosphorylated-PhoB, resulting in downstream expression of GFP. When a high concentration of extracellular phosphate ions is present, PhoR will dephosphorylate PhoB, and therefore inactivating it, and repressing the PhoB promoter for GFP expression. [[Image:Phosphate1.png|thumb|center|500px|Figure 1: PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP.]]
+====Improvement over previous iGEM part [https://parts.igem.org/Part:BBa_K116404 BBa_K116404 (NYMU Taipei 2008)]====
+The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbone and subsequently characterized in <i>E. coli</i> MG 1655. These cells were grown in LB medium before resuspension in MOPS medium (a minimal nutrient medium) for characterization with a micro-plate reader. Varying concentrations of phosphate ions from 0 to 1000 uM were added and GFP expression was monitored.
+By replacing the weaker RBS BBa_B0032 of the original part with a stronger RBS BBa_B0034, we have successfully constructed an improved phosphate sensor-GFP reporter. Our part shows, on average, a 40-fold increase in GFP expression (Figure 3) when compared to the previous version of the construct (http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate). The original phosphate construct is also insensitive to high phosphate concentrations above 50 µM where similar levels of GFP expression are observed (Figure 4). Unlike the previous construct, our improved phosphate construct is much more sensitive to various phosphate concentrations from 0 to 1000 µM, particularly at phosphate concentrations above 50 µM.
+[[Image:Phosphate5.png|thumb|center|800px|Figure 2: Side by side comparison of our improved construct Bba_K2447000 & the original construct BBa_K116404. Our proposed part exhibited greater GFP expression at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50 uM.]]
+[[Image:Phosphate6.png|thumb|center|800px|Figure 3: Our improved phosphate construct is much more sensitive to phosphate concentrations above 50 uM, unlike the original construct.]]
+[[Image:Phosphate7.png|thumb|center|800px|Figure 4: GFP expression by the original phosphate construct designed by the Taiwanese team.]]
-====Improvement over previous iGEM part BBa_K116404 (NYMUTaipei 2008)====
-The fluorescence of purified GFPmut3B was calibrated in the [[Chassis/Cell-Free_Systems|cell-free chassis]]. The derived [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.3 calilbration curve]allows the determination of the concentration of GFPmut3b in the cell-free chassis. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.3 Detailed protocols]for generating the calibration curve are available. Other calibration curves for are also available on the results page.[[Image:Phosphate2.png|thumb|center|800px|PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP]]
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-[[Image:Phosphate3.png|thumb|center|800px|PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP]]