Difference between revisions of "Part:BBa K2447000"

Revision as of 08:09, 24 October 2017

Extracellular phosphate sensor with GFP reporter

PhoB and PhoR proteins are part of the Pho regulon 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 PhoB, and therefore inactivating it, and repressing PhoB promoter for downstream expression of GFP.

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 transferring into 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, 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 in yielding GFP p. Our proposed part exhibited greater GFP expression at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50uM.

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

Figure 4: 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|Figure 1: 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 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 PhoB, and therefore inactivating it, and repressing PhoB promoter for downstream expression of GFP. [[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 backbones and subsequently characterised in E.coli MG 1655. These cells were grown in LB medium before transferring into 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 productions were monitored.
+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 transferring into 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 32 (as utilised by the Taiwanese team) with a stronger binding affinity RBS 34 as proposed by iGEM NUS team 2017, we have elucidated much stronger GFP expression and also improved the sensitivity of the part to varying concentrations of phosphate ions. Previously, the Taiwanese part [http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate had reported]much lower levels of GFP productions for all phosphate concentrations. Our improved construct had yielded on average 40 fold increase (Figure 2) in GFP expression when compared to the original construct. In addition, our proposed phosphate sensor is more sensitive (Figure 3) at higher levels of phosphate concentrations, and able to yield different levels of repressed GFP productions. On the other hand, the Taiwanese construct remains not sensitive to high amount of phosphate concentrations, yielding similar level of repressed GFP productions (Figure 4) when the phosphate concentrations is above 50 uM.
+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, 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 in yielding GFP p. Our proposed part exhibited greater GFP expressions at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50uM.]]
+[[Image:Phosphate5.png|thumb|center|800px|Figure 2: Side by side comparison of our improved construct Bba_K2447000 & the original construct BBa_K116404 in yielding GFP p. Our proposed part exhibited greater GFP expression at every phosphate concentration and exhibited much higher sensitivity to phosphate levels above 50uM.]]