Difference between revisions of "Part:BBa K629013"

 
 
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<partinfo>BBa_K629013 short</partinfo>
 
<partinfo>BBa_K629013 short</partinfo>
  
 
used to test the TrkD when expression is controlled by a strong promoter.
 
used to test the TrkD when expression is controlled by a strong promoter.
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== Background ==
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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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== 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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[[Image:TrkD.jpg]]
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== Detailed Paramers in Result Report ==
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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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2) 37℃ for 4h, 200r.
 +
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3) OD: 0.6~1.0
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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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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 08:31, 9 October 2011

T7 promoter + lac operator + RBS + trkD

used to test the TrkD when expression is controlled by a strong promoter.


Background

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.


Expression in E. Coli

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.

Note: Because the promoter is Plac here, the expression has a background, which means control also expresses some TrkD without induction of IPTG.

TrkD.jpg


Detailed Paramers in Result Report

One of the test reports is below. If you need more, please contact me.

Before test, our samples have been treated with below methods.

1) Induction of 100mM IPTG

2) 37℃ for 4h, 200r.

3) OD: 0.6~1.0

4）400nmol cesium orginally

5) use ddH2O to clean the bacterial over 3 times.

Cs Report.jpg


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 61
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 346
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 61
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 61
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1421