Difference between revisions of "Part:BBa K2505001"
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==Characterization==
 
==Characterization==
 
We tested AHK4 protein receives iP and induces histidine kinase phosphorylation in <i>E.coli</i> cell. Our objective was to characterize the function of AHK4 protein.  
 
We tested AHK4 protein receives iP and induces histidine kinase phosphorylation in <i>E.coli</i> cell. Our objective was to characterize the function of AHK4 protein.  
 +
The purpose of experiments on this page is to confirm that AHK4 can receive iP, a signal molecule produced by human cells, and AHK4→RcsD→RscB→cps pathyway will be activated in turn. To see the activation of the pathway, we used <i>E.coli</i> KMI002 strain as a carrier of AHK4. This KMI002 possesses cps::lacZ fusion gene and the activation of AHK4→RcsD→RscB→cps::lacZ pathway can be observed through the activity of β-galactosidase.
 +
As a qualitative experiment, we observed it if AKH4 carrying cells develop blue color under the existence of iP and X-gal on agar plates.
 +
As a quantitative experiment, we cultured E. coli with various concentrations of iP in liquid medium and measured β-galactosidase activity by using ONPG.
  
 
===Materials & methods===
 
===Materials & methods===

Revision as of 13:14, 27 October 2017

pBad/araC-rbs-ahk4

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1294
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 2375
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI site found at 1542
    Illegal SapI.rc site found at 3177


This part produces AHK4, a receptor of iP(isopentenyl adenine: kind of cytokinin). This part was introduced to E.coli(KMI002) and sensed iP synthesized by human cell. The gene(ahk4) is derived from A.thaliana and optimized for E.coli codon in reference to the codon usage. The AHK4 protein is transmembrane protein and toxic for E.coli cell. Thus, We regulated expression of AHK4 by PBAD/araC: L-arabinose operon(tight transcriptionaly regulation system).

Characterization

We tested AHK4 protein receives iP and induces histidine kinase phosphorylation in E.coli cell. Our objective was to characterize the function of AHK4 protein. The purpose of experiments on this page is to confirm that AHK4 can receive iP, a signal molecule produced by human cells, and AHK4→RcsD→RscB→cps pathyway will be activated in turn. To see the activation of the pathway, we used E.coli KMI002 strain as a carrier of AHK4. This KMI002 possesses cps::lacZ fusion gene and the activation of AHK4→RcsD→RscB→cps::lacZ pathway can be observed through the activity of β-galactosidase. As a qualitative experiment, we observed it if AKH4 carrying cells develop blue color under the existence of iP and X-gal on agar plates. As a quantitative experiment, we cultured E. coli with various concentrations of iP in liquid medium and measured β-galactosidase activity by using ONPG.

Materials & methods