Difference between revisions of "Part:BBa K2680550"
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__NOTOC__ | __NOTOC__ | ||
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<partinfo>BBa_K2680550 short</partinfo> | <partinfo>BBa_K2680550 short</partinfo> | ||
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de-GFP ssra is a destabilized GFP created by "fusing amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP) <sup>1</sup> This destabilized GFP is fitted with an ssrA degradation tag. | de-GFP ssra is a destabilized GFP created by "fusing amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP) <sup>1</sup> This destabilized GFP is fitted with an ssrA degradation tag. | ||
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===References=== | ===References=== | ||
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1. Li‡, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., & Huang, C. F. (1998, December 25). Xianqiang Li. Retrieved from http://www.jbc.org/content/273/52/34970.full.html | 1. Li‡, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., & Huang, C. F. (1998, December 25). Xianqiang Li. Retrieved from http://www.jbc.org/content/273/52/34970.full.html | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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<partinfo>BBa_K2680550 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2680550 SequenceAndFeatures</partinfo> | ||
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'''Group:''' Team ZJUT-China 2021 <br> | '''Group:''' Team ZJUT-China 2021 <br> | ||
'''Author:''' Lianjie Sha and Xia Yao <br> | '''Author:''' Lianjie Sha and Xia Yao <br> | ||
− | '''Summary:''' | + | '''Summary:''' According to the lectures, we learned that the degradation rate of eGFP-ssrA could be measured. In the cell-free system, protein degradation by clpXP is described by a zeroth order chemical kinetic.In protein substrates (eGFP), ClpX recognizes ssrA--a specific C-terminal degradation tag, proceeds to unfold stable tertiary structure in the protein, and then spools or translocates the unfolded polypeptide chain into a sequestered proteolytic compartment in ClpP for degradation into small peptide fragments. |
− | + | As part of iGEM18_William_and_Mary, ZJUT-China assessed the degradation rate of different eGFP [https://parts.igem.org/Part:BBa_K2680550 (Part:BBa_K2680550)]by adding different concentrate of plasmid P70-ClpXP [https://parts.igem.org/Part:BBa_K3885203 (Part:BBa_K3885203)]based on the reference. We hope this will enhance further modeling and experiments. | |
===Methodology=== | ===Methodology=== | ||
− | + | There are two methods to express clpxp protein: co-expression and pre-expression.Accelerated protein degradation can be achieved by co-expression of P70a-ClpXP, by adding protein to a cell-free system pre-incubated with P70a-ClpXP for an hour or by adding dilutions of pre-expressed ClpXP (P70a-ClpXP, 3nM). Different methods can provide different rates of protein degradation, ranging from 9.3 nM/min to 250 nM/min. By expressing ClpXP, protein synthesis can be adjusted to an appropriate rate[1]. | |
===Results=== | ===Results=== | ||
− | + | <html> | |
+ | <style> | ||
+ | .flex{ | ||
+ | display: flex; | ||
+ | align-items: center; | ||
+ | justify-content: space-evenly; | ||
+ | } | ||
+ | </style> | ||
+ | <div class="flex" style="margin: 0 auto; width: 100%;"> | ||
+ | <div style="border: 1px solid #000;width: 50%; background-color: #f9f9f9;"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/e/ea/268.png" width=95% style="display: block;margin: 10px auto;"/> | ||
+ | <p style="text-align: center;">Figure 1 Degradation rate of deGFP upon different expression methods of ClpXP. </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | The degradation rate of eGFP-ssrA only using the endogenous ClpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-eGFP-ssrA (5μM) to the Cell-Free system. | ||
===Analysis === | ===Analysis === | ||
+ | As shown in the figure above, the higher the concentration of ClpXP, the faster the degradation of eGFP-ssrA. Meanwhile, according to the table, when ClpXP with the same concentration was added, eGFP degradation rate in pre-expression was from 6.5 nM/min to 256 nM/min, while in co-expression, eGFP degradation rate was from 9.3 nM/min to 128 nM/min. It can be concluded that pre-expression is more conducive to eGFP protein degradation than co-expression. | ||
+ | ===Reference=== | ||
+ | [1] Garamella J, Marshall R, Rustad M, et al. The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology[J]. ACS synthetic biology, 2016, 5(4): 344-355. | ||
Latest revision as of 03:00, 20 October 2021
deGFP-ssra
de-GFP ssra is a destabilized GFP created by "fusing amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP) 1 This destabilized GFP is fitted with an ssrA degradation tag.
References
1. Li‡, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., & Huang, C. F. (1998, December 25). Xianqiang Li. Retrieved from http://www.jbc.org/content/273/52/34970.full.html
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 47
Illegal BsaI.rc site found at 771
Contribution by Team ZJUT-China 2021
Group: Team ZJUT-China 2021
Author: Lianjie Sha and Xia Yao
Summary: According to the lectures, we learned that the degradation rate of eGFP-ssrA could be measured. In the cell-free system, protein degradation by clpXP is described by a zeroth order chemical kinetic.In protein substrates (eGFP), ClpX recognizes ssrA--a specific C-terminal degradation tag, proceeds to unfold stable tertiary structure in the protein, and then spools or translocates the unfolded polypeptide chain into a sequestered proteolytic compartment in ClpP for degradation into small peptide fragments.
As part of iGEM18_William_and_Mary, ZJUT-China assessed the degradation rate of different eGFP (Part:BBa_K2680550)by adding different concentrate of plasmid P70-ClpXP (Part:BBa_K3885203)based on the reference. We hope this will enhance further modeling and experiments.
Methodology
There are two methods to express clpxp protein: co-expression and pre-expression.Accelerated protein degradation can be achieved by co-expression of P70a-ClpXP, by adding protein to a cell-free system pre-incubated with P70a-ClpXP for an hour or by adding dilutions of pre-expressed ClpXP (P70a-ClpXP, 3nM). Different methods can provide different rates of protein degradation, ranging from 9.3 nM/min to 250 nM/min. By expressing ClpXP, protein synthesis can be adjusted to an appropriate rate[1].
Results
Figure 1 Degradation rate of deGFP upon different expression methods of ClpXP.
Analysis
As shown in the figure above, the higher the concentration of ClpXP, the faster the degradation of eGFP-ssrA. Meanwhile, according to the table, when ClpXP with the same concentration was added, eGFP degradation rate in pre-expression was from 6.5 nM/min to 256 nM/min, while in co-expression, eGFP degradation rate was from 9.3 nM/min to 128 nM/min. It can be concluded that pre-expression is more conducive to eGFP protein degradation than co-expression.
Reference
[1] Garamella J, Marshall R, Rustad M, et al. The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology[J]. ACS synthetic biology, 2016, 5(4): 344-355.