Difference between revisions of "Part:BBa K2680550"

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===Results===
 
===Results===
 
Group1: Pre-expression for an hour
 
Group1: Pre-expression for an hour
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{|class="wikitable"
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<html>
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|ClpXP [nM]
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<table border="1" cellspacing="0" cellpadding="0" style="border: 1px solid #000;">
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|eGFP degradation rate [nM/min]
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        <tr>
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|-
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            <td>ClpXP [nM]</td>
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|0
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            <td>eGFP degradation rate [nM/min]</td>
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|6.51±1.25
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        </tr>
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|-
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        <tr>
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|0.2
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            <td>0</td>
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|28.04±3.87
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            <td>6.51±1.25</td>
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|-
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        </tr>
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|0.4
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        <tr>
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|48.24±8.06
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            <td>0.2</td>
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|-
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            <td>28.04±3.87</td>
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|1
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        </tr>
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|88.32±17.71
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        <tr>
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|-
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            <td>0.4</td>
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|2
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            <td>48.24±8.06</td>
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|159.13±21.92
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        </tr>
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|-
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        <tr>
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|6
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            <td>1</td>
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|256.07±38.24
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            <td>88.32±17.71</td>
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|}
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        </tr>
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<html><img src="https://static.igem.org/mediawiki/parts/8/8c/T--ZJUT-China--pre_expression_1.png" width="100"></html>
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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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        <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="100">
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</html>
 
Group2：Co-expression
 
Group2：Co-expression
 
左边表格，右边图片
 
左边表格，右边图片

Revision as of 13:18, 10 October 2021

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 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 (Part:BBa_K3885203) based on the reference. We hope it will support more help on modelling and further 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

Group1: Pre-expression for an hour

ClpXP [nM] eGFP degradation rate [nM/min]
0 6.51±1.25
0.2 28.04±3.87
0.4 48.24±8.06
1 88.32±17.71
2 159.13±21.92
6 256.07±38.24
Group2：Co-expression 左边表格，右边图片

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.

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.