Difference between revisions of "Part:BBa K1051258"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1051258 short</partinfo>
 
<partinfo>BBa_K1051258 short</partinfo>
  
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Construct the final pathway with M0051
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<h3>Purpose</h3>
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Construct the measurement pathway of K1051207.
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<br />
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<h3>Principle</h3>
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'''SsrA degradation tag'''. In E. <i>coli</i>, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of M0051 to construnt this K1051207.<br />
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We constructed the measurement pathway K1051258(contains J04500, K1051000 and K1051207) to test the rates of degradation of tagged proteins. J04450 was used as positive control because of the same promoter and fluorescent protein.
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<br />
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
  
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1051258 SequenceAndFeatures</partinfo>
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<h3>Measurement</h3>
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https://static.igem.org/mediawiki/2013/b/b0/Curve_ecdeg.png
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The growth curve of E. <i>coli</i>.
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https://static.igem.org/mediawiki/2013/e/e9/Degladder.jpg
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From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing.
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https://static.igem.org/mediawiki/2013/6/63/Flo.jpg
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https://static.igem.org/mediawiki/igem.org/8/86/Flo2.jpg
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The test results of BBa_K1051258. A:No exciting lights; B. Powerful exciting lights; C. Weak exciting lights.
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In picture, there are only obvious lights in the picture B, indicated the degradation rates are working
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https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg
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The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes
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<br />
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<h3>Sequence and Features</h3>
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<partinfo>BBa_K1051257 SequenceAndFeatures</partinfo>
  
  
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<partinfo>BBa_K1051258 parameters</partinfo>
 
<partinfo>BBa_K1051258 parameters</partinfo>
 
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<h3>References</h3>
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[1]McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701.<br />
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[2]Flynn et al 2003. Mol. Cell. 11: 671. Flynn et al. 2001. PNAS 98(19): 10584. Anderson et al 1998. App. Env. Microbiol. 64(6):2240

Revision as of 17:20, 27 September 2013

Construct the measurement pathway of K1051207.

Purpose

Construct the measurement pathway of K1051207.

Principle

SsrA degradation tag. In E. coli, the adaptor SspB tethers ssrAtagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. Which means, a variation of the WT SsrA tag sequence will accelerate the degradation of proteins when fused to their C-terminal. Thus the degradation rates are dependent on concentration of proteases and binding mediators. In order to fuse degradation tags freely on the C-terminal of protein, we add TAATAA to the tail of M0051 to construnt this K1051207.
We constructed the measurement pathway K1051258(contains J04500, K1051000 and K1051207) to test the rates of degradation of tagged proteins. J04450 was used as positive control because of the same promoter and fluorescent protein.

Measurement

Curve_ecdeg.png The growth curve of E. coli. Degladder.jpg From right to left, the negative control,BBa_K1051257, BBa_K1051258, BBa_K1051259, J04450 as Positive Control. As the pictures showed, the lights of RFP within three degradation tags are decreasing. Flo.jpg Flo2.jpg The test results of BBa_K1051258. A:No exciting lights; B. Powerful exciting lights; C. Weak exciting lights. In picture, there are only obvious lights in the picture B, indicated the degradation rates are working Degmicro.jpg The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 781
    Illegal AgeI site found at 893
  • 1000
    COMPATIBLE WITH RFC[1000]



References

[1]McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701.
[2]Flynn et al 2003. Mol. Cell. 11: 671. Flynn et al. 2001. PNAS 98(19): 10584. Anderson et al 1998. App. Env. Microbiol. 64(6):2240