Difference between revisions of "Part:BBa K629005"

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<partinfo>BBa_K629005 short</partinfo>
 
<partinfo>BBa_K629005 short</partinfo>
  
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'''
 
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== Background ==
 
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'''
 
  
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Escherichia coli cells which contain a functional Kup (formerly TrkD) system took up Cs' with a moderate rate and affinity. Kup is a separate K+ uptake system with relatively little discrimination in the transport of the cations K+, Rb+, and Cs'. Regardless of the presence or absence of Kup, K+-replete cells took up Cs'primarily by a very low affinity mode, proportional to the ratio of the Cs' and K+ concentrations in the medium. In our project, trkD is used to collect Cs-137 when recN is activated by radiation and start the transcription of trkD,in order to absorb this kind of radioactive source like Cs-137.
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== Introduction and Background ==
  
  
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Protein TrkD (belongs to Kup system) is responsible for the low-affinity transport of potassium into the cell, but with high affinity to transport cesium. In contrast to Trk or Kdp, the Kup system does not strongly distinguish between the alkali cations K+, Rb+, or even Cs+. When both of Cs+ and K+ exist, the uptake of Cs+ has inhibition of that of K+. And among several proteins in Kup system, TrkD has the best effect of uptake of Cs+. Accordingly, after E. Coli approached Cs+, the expression of TrkD will help our E. Coli to absorb Cs-137.
  
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== Expression in ''E. Coli'' ==
 
  
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Before we started our functional test of TrkD, we have utilized western blot to determine whether there are enough soluble proteins in E. Coli. With 6×His tag on pET-32a, the result shows that TrkD has more expression with induction of IPTG.
 
  
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Note: Because the promoter is Plac here, the expression has a background, which means control also expresses some TrkD without induction of IPTG.
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== Methods to quantify the efficiency ==
  
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[[Image:TrkD.jpg]]
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The research about this protein are relatively few and old. It's difficult to contact with original authors to get the details in the experiments. As a result, we need to verify the value of TrkD in our project.
  
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Then, we first construct gene trkD on plasmid pET-32a to test the useful expression of TrkD. After the western-blot test, we construct it on pSB1C3 with promoter T7 and operator lac (see the detail in BBa_K629013), which can be induced by IPTG.
  
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== Detailed Paramers in Result Report ==
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The test of function of TrkD is not easy, so we sent the sample to the test center in Guangzhou to help us.
  
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One of the test reports is below. If you need more, please contact me.
 
  
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Before test, our samples have been treated with below methods.
 
  
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1) Induction of 100mM IPTG
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== Results ==
  
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2) 37℃ for 4h, 200r.
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The results shown below:
  
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3) OD: 0.6~1.0
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[[Image:Cesium_absorption.jpg]]
  
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4）400nmol cesium orginally
 
  
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5) use ddH2O to clean the bacterial over 3 times.
 
  
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[[Image:Cs Report.jpg]]
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== Reference ==
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D. Bossemeyer et al., Journal of Bacteriology. 171, 2219 (1989).
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Latest revision as of 05:02, 5 November 2011

trkD, a functional Kup (formerly TrkD) system took up Cs+ with a moderate rate and affinity


Introduction and Background

Protein TrkD (belongs to Kup system) is responsible for the low-affinity transport of potassium into the cell, but with high affinity to transport cesium. In contrast to Trk or Kdp, the Kup system does not strongly distinguish between the alkali cations K+, Rb+, or even Cs+. When both of Cs+ and K+ exist, the uptake of Cs+ has inhibition of that of K+. And among several proteins in Kup system, TrkD has the best effect of uptake of Cs+. Accordingly, after E. Coli approached Cs+, the expression of TrkD will help our E. Coli to absorb Cs-137.


Methods to quantify the efficiency

The research about this protein are relatively few and old. It's difficult to contact with original authors to get the details in the experiments. As a result, we need to verify the value of TrkD in our project.

Then, we first construct gene trkD on plasmid pET-32a to test the useful expression of TrkD. After the western-blot test, we construct it on pSB1C3 with promoter T7 and operator lac (see the detail in BBa_K629013), which can be induced by IPTG.

The test of function of TrkD is not easy, so we sent the sample to the test center in Guangzhou to help us.


Results

The results shown below:

Cesium absorption.jpg


Reference

D. Bossemeyer et al., Journal of Bacteriology. 171, 2219 (1989).



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 245
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1320