Difference between revisions of "Part:BBa K2680550"

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__NOTOC__
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<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.
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===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
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2680550 SequenceAndFeatures</partinfo>
  
 
=='''Contribution by Team ZJUT-China 2021'''==
 
=='''Contribution by Team ZJUT-China 2021'''==
 
'''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>
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'''Summary:''' According to the lectures, we learned that the degradation rate of eGFP-ssrA could be measured. In cell-free system, protein degradation by clpXP is described by a zeroth order chemical kinetic,and clpxp protein can recognize the protein with ssrA tag, so it is useful to add clpxp degrading the degfp. This year,on the basis work of iGEM18_William_and_Mary, ZJUT-China measured the different eGFP degradation rate by adding different concentrate of plasmid P70-clpxp [https://parts.igem.org/Part:BBa_K3885203 (Part:BBa_K3885203)] based on the reference. We hope it will support more help on modelling and further experiments.
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'''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.
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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===
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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]
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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===
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Group1: Pre-expression for an hour
 
 
<html>
 
<html>
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<table border="1" cellspacing="0" cellpadding="0" style="display: inline;">
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<style>
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        <tr>
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.flex{
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            <td>ClpXP [nM]</td>
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    display: flex;
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            <td>eGFP degradation rate [nM/min]</td>
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    align-items: center;
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        </tr>
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    justify-content: space-evenly;
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        <tr>
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}
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            <td>0</td>
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</style>
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            <td>6.51±1.25</td>
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<div class="flex" style="margin: 0 auto; width: 100%;">
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        </tr>
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<div style="border: 1px solid #000;width: 50%; background-color: #f9f9f9;">
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        <tr>
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<img src="https://static.igem.org/mediawiki/parts/e/ea/268.png" width=95% style="display: block;margin: 10px auto;"/>
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            <td>0.2</td>
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<p style="text-align: center;">Figure 1 Degradation rate of deGFP upon different expression methods of ClpXP.  </p>
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            <td>28.04±3.87</td>
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</div>
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        </tr>
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</div>
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            <td>0.4</td>
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            <td>48.24±8.06</td>
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        </tr>
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        <tr>
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            <td>1</td>
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            <td>88.32±17.71</td>
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        </tr>
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        <tr>
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            <td>2</td>
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            <td>159.13±21.92</td>
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        </tr>
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            <td>6</td>
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            <td>256.07±38.24</td>
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        </tr>
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    </table>
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<img src="https://static.igem.org/mediawiki/parts/8/8c/T--ZJUT-China--pre_expression_1.png" width="250"><br>
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</html>
 
</html>
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Group2：Co-expression
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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.
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左边表格，右边图片
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Degradation rate of deGFP upon different expression methods of clpXP. The degradation rate of deGFP-ssra only using the endogenous clpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-GFP-ssrA (5μM) to the cell-free system.
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===Analysis ===
 
===Analysis ===
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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.
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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===
 
===Reference===

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



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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.

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

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.