Difference between revisions of "Part:BBa K3885203"
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<partinfo>BBa_K3885203 short</partinfo> | <partinfo>BBa_K3885203 short</partinfo> | ||
− | + | The E. coli complex ClpXP, a protease from the AAA+ family, is an ideal system to achieve targeted protein degradation in Cell-Free reactions by adding, either to the N-terminus or C-terminus, a specifific degron typically 10−12 amino acids long.56 In Cell-Free systems,46 as well as in E. coli, 57 protein degradation by ClpXP is a zeroth order reaction with constant rate. <br> | |
− | The E. coli complex ClpXP, a protease from the AAA+ family, is an ideal system to achieve targeted protein degradation in Cell-Free reactions by adding, either to the N-terminus or C-terminus, a specifific degron typically 10−12 amino acids long.56 In Cell-Free systems,46 as well as in E. coli, 57 protein degradation by ClpXP is a zeroth order reaction with constant rate. | + | Under the influence of the P70 promoter, ClpXP protein can be expressed.The expression of ClpXP in Cell-Free system can improve the degradation rate of protein with ssrA degradation label. |
+ | ===Usage and Biology=== | ||
+ | <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/2/28/337-1.png" width=95% height=70% style="display: block;margin: 10px auto;"/> | ||
+ | <p style="text-align: center:">Figure 1. Schematic of hydrolysis by ClpXP protein. </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | To ensure the normal operation of the Cell-Free system, an adjustable protein degradation mechanism is crucial. It is degraded by ClpXPAAA+ protease in Escherichia coli . The protein must contain a degradation tag in order to be recognized by our cell-free system .<br> | ||
+ | |||
+ | </html> | ||
+ | |||
+ | ===Characterization=== | ||
+ | <html> | ||
+ | <style> | ||
+ | .flex{ | ||
+ | display: flex; | ||
+ | align-items: center; | ||
+ | justify-content: space-evenly; | ||
+ | } | ||
+ | </style> | ||
+ | <div style="margin: 0 auto; width: 100%;"> | ||
+ | <div style="border: 1px solid #000;width: 80%; background-color: #f9f9f9;margin: 0 auto;"> | ||
+ | <div class="flex" style="width:100%;"> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/1/18/Gai11.png" width=90% style=" margin: 10px auto;"/> | ||
+ | </div> | ||
+ | <p style="text-align: center:">Figure 2. Schematic of gene circuits and kinetics of tetR inhibition in the Cell-Free system.<br> | ||
+ | (A) The gene circuits contains three parts, sigma 28 activates promoter P28 to express tet repressor with ssrA tag, which inhibits the expression of deGFP. <br> | ||
+ | (B) The gene circuits contains an additional part P70-ClpXP that makes the tetR repressor degrade, and degfp expresses.<br> | ||
+ | (C) Group 1 carried tetR without ssrA and ClpXP; Group 2 carried tetR with ssrA; Group 3 didn't carry tetR as positive control; Group 4 carried tetR with ssrA and ClpXP in comparison with Group 1.</p> | ||
+ | </div> | ||
+ | |||
+ | </div><br> | ||
+ | In Figure 2. C, the highest red line ( Group 3) contains only P70a-σ28 and P28-tetO-deGFP plasmids, thus expressing deGFP with high fluorescence intensity and serving as a positive control. The second highest purple line ( Group 4) with P70a-ClpXP can degrade tetR repressor with ssrA tag, eliminate its inhibition of deGFP expression in downstream of the gene circuits, and increase fluorescence intensity. The green line at the bottom of Figure 2. C ( Group 1) indicates that the tetR repressor without ssrA tag cannot be degraded by ClpXP protein, in contrast to Group 4, indicating that the ssrA degradation tag is functional. The blue line ( Group 2) is at the bottom of Figure 2. C showed low fluorescence intensity, which was used as a negative control to indicate that the addition of tag did not affect the inhibition of tetR. <br> | ||
+ | <strong>Therefore, we gave the tetR a new function and improved this part.</strong> | ||
+ | <br> | ||
+ | </html> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Latest revision as of 18:04, 21 October 2021
P70-ClpXP
The E. coli complex ClpXP, a protease from the AAA+ family, is an ideal system to achieve targeted protein degradation in Cell-Free reactions by adding, either to the N-terminus or C-terminus, a specifific degron typically 10−12 amino acids long.56 In Cell-Free systems,46 as well as in E. coli, 57 protein degradation by ClpXP is a zeroth order reaction with constant rate.
Under the influence of the P70 promoter, ClpXP protein can be expressed.The expression of ClpXP in Cell-Free system can improve the degradation rate of protein with ssrA degradation label.
Usage and Biology
![](https://static.igem.org/mediawiki/parts/2/28/337-1.png)
Figure 1. Schematic of hydrolysis by ClpXP protein.
Characterization
![](https://static.igem.org/mediawiki/parts/1/18/Gai11.png)
Figure 2. Schematic of gene circuits and kinetics of tetR inhibition in the Cell-Free system.
(A) The gene circuits contains three parts, sigma 28 activates promoter P28 to express tet repressor with ssrA tag, which inhibits the expression of deGFP.
(B) The gene circuits contains an additional part P70-ClpXP that makes the tetR repressor degrade, and degfp expresses.
(C) Group 1 carried tetR without ssrA and ClpXP; Group 2 carried tetR with ssrA; Group 3 didn't carry tetR as positive control; Group 4 carried tetR with ssrA and ClpXP in comparison with Group 1.
In Figure 2. C, the highest red line ( Group 3) contains only P70a-σ28 and P28-tetO-deGFP plasmids, thus expressing deGFP with high fluorescence intensity and serving as a positive control. The second highest purple line ( Group 4) with P70a-ClpXP can degrade tetR repressor with ssrA tag, eliminate its inhibition of deGFP expression in downstream of the gene circuits, and increase fluorescence intensity. The green line at the bottom of Figure 2. C ( Group 1) indicates that the tetR repressor without ssrA tag cannot be degraded by ClpXP protein, in contrast to Group 4, indicating that the ssrA degradation tag is functional. The blue line ( Group 2) is at the bottom of Figure 2. C showed low fluorescence intensity, which was used as a negative control to indicate that the addition of tag did not affect the inhibition of tetR.
Therefore, we gave the tetR a new function and improved this part.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 448
Illegal EcoRI site found at 1510
Illegal EcoRI site found at 1855 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 448
Illegal EcoRI site found at 1510
Illegal EcoRI site found at 1855 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 448
Illegal EcoRI site found at 1510
Illegal EcoRI site found at 1855
Illegal BglII site found at 1689 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 448
Illegal EcoRI site found at 1510
Illegal EcoRI site found at 1855 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 448
Illegal EcoRI site found at 1510
Illegal EcoRI site found at 1855
Illegal AgeI site found at 1171
Illegal AgeI site found at 1909 - 1000COMPATIBLE WITH RFC[1000]