Difference between revisions of "Part:BBa K1051206"
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<h3>Principle</h3> | <h3>Principle</h3> | ||
| − | '''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 M0050 to construnt this part.<br /> | + | '''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 M0050 to construnt this part.<br /> |
We constructed the measurement pathway of tag K1051257(contains J04500, K1051000, K1051206) to test the rates of degradation of tagged proteins. J04450 was used as positive control because of the same promoter and fluorescent protein. | We constructed the measurement pathway of tag K1051257(contains J04500, K1051000, K1051206) 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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| + | <h3>Measurement</h3> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/b/b0/Curve_ecdeg.png"> | ||
| + | The growth curve of E. <i>coli</i>. | ||
| + | <img src="https://static.igem.org/mediawiki/2013/e/e9/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. | ||
| + | <img src="https://static.igem.org/mediawiki/2013/6/63/Flo.jpg"> | ||
| + | <img scr="https://static.igem.org/mediawiki/igem.org/8/86/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 | ||
| + | <img src="https://static.igem.org/mediawiki/2013/a/a8/Degmicro.jpg"> | ||
| + | The test results of BBa_K1051258 in chip. A,LB medium,O minuts; B, IPTG medium,9minutes; C,IPTG medium, 15 minutes | ||
| + | <br /> | ||
<h3>Sequence and Features</h3> | <h3>Sequence and Features</h3> | ||
Revision as of 16:35, 27 September 2013
The degradation tag in E. coil ,M0050 with TAATAA.
Purpose
SsrA degradation tag in E. coli, add TAATAA to C-terminal of Biobrick M0050.
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 M0050 to construnt this part.
We constructed the measurement pathway of tag K1051257(contains J04500, K1051000, K1051206) to test the rates of degradation of tagged proteins. J04450 was used as positive control because of the same promoter and fluorescent protein.
Measurement
<img src="
">
The growth curve of E. coli.
<img src="
">
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.
<img src="
">
<img scr="
">
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
<img src="
">
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1]McGinness, Baker, Sauer. 2006. Mol. Cell. 22:701.</h3> [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