Difference between revisions of "Part:BBa K2447000"

Revision as of 13:39, 12 October 2017

Extracellular phosphate sensor with GFP reporter

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.

PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP

Improvement over previous iGEM part BBa_K116404 (NYMUTaipei 2008)

The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbones and subsequently characterised in E.coli MG1656. These cells are grown in MOPS medium (a minimal nutrient medium) and varying concentrations of phosphate ions from 0 to 1000 uM are added.

PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP

asdsadsadsadasasdasdsadadas

PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP

The two parts (Bba. ...) listed below are inserted into e8k backbones and subsequently characterised in E.coli MG1656. These cells are grown in MOPS medium (a minimal nutrient medium) and varying concentrations of phosphate ions from 0 to 1000 uM are added.

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 sensivity of the part to varying concentrations of phosphate ions. Previously, the Taiwanese part had reported similar level of repressed GFP productions for phosphate concentrations above 40uM. On the other hand, our part is sensitive to phosphate concentration above 40 uM, elucidating differential GFP expressions when placed in different phosphate level.

figure 2: side by side comparison of construct Bba.... upon expressing GFP/OD production as a percentage. our proposed part exhibited greater sensitivity to phosphate levels above 40uM. figure 3: Our proposed phosphate construct, sensitive to phosphate concentrations from 0 to 1000 uM.

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

@@ Line 5: / Line 5: @@
 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]]
-====Improvement over previous iGEM part BBa_K116404 (NYMUTaipei 2008)====
+====Improvement over previous iGEM part [https://parts.igem.org/Part:BBa_K116404 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]]
+The two parts (Bba_K2447000 & BBa_K116404) listed below are inserted into pBbE2k backbones and subsequently characterised in E.coli MG1656. These cells are grown in MOPS medium (a minimal nutrient medium) and varying concentrations of phosphate ions from 0 to 1000 uM are added.
+[[Image:Phosphate2.png|thumb|center|800px|PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP]]
 asdsadsadsadasasdasdsadadas
 [[Image:Phosphate3.png|thumb|center|800px|PhoR and PhoB proteins work in tandem to control promoter PhoB and consequential downstream expression of GFP]]
+The two parts (Bba. ...) listed below are inserted into e8k backbones and subsequently characterised in E.coli MG1656. These cells are grown in MOPS medium (a minimal nutrient medium) and varying concentrations of phosphate ions from 0 to 1000 uM are added.
+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 sensivity of the part to varying concentrations of phosphate ions. Previously, the Taiwanese part had reported similar level of repressed GFP productions for phosphate concentrations above 40uM. On the other hand, our part is sensitive to phosphate concentration above 40 uM, elucidating differential GFP expressions when placed in different phosphate level.
+figure 2: side by side comparison of construct Bba.... upon expressing GFP/OD production as a percentage. our proposed part exhibited greater sensitivity to phosphate levels above 40uM.
+figure 3: Our proposed phosphate construct, sensitive to phosphate concentrations from 0 to 1000 uM.
 <!-- Add more about the biology of this part here