Difference between revisions of "Part:BBa K337028"
 
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The TetR binds to the Tet Operator and by that causes knockdown auf gene expression. (BB-2, RFC12).
 
The TetR binds to the Tet Operator and by that causes knockdown auf gene expression. (BB-2, RFC12).
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As the <strong>improvement</strong> of the 10_Heidelberg registered part (<a href="https://parts.igem.org/Part:BBa_K337028">BBa_K337028</a>: tet Repressor), we, ZJUT-China 2021 newly registered part<a href="https://parts.igem.org/Part:BBa_K3885126">BBa_K3885126.</a>  through <strong>adding a degradation tag ssrA</strong> to the 3' end of the tetR (Figure 1), endowing the tetR <strong>a novel function—degradation.</strong>
 
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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>  
 
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>  
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As the improvement of the 10_Heidelberg registered part (<a href="https://parts.igem.org/Part:BBa_K3885311">BBa_K337028</a>: tet Repressor)  , the degradation tag ssrA is added to the 3' end of the tetR (Figure 1) , hence the newly registered part<a href="https://parts.igem.org/Part:BBa_K3885311">BBa_K3885126.</a> <br>
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<img src="https://static.igem.org/mediawiki/parts/d/d0/337-2.png" img src="https://static.igem.org/mediawiki/parts/d/da/337-3.png" width=95% height=70% style="display: block;margin: 10px auto; "/>
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<img src="https://static.igem.org/mediawiki/parts/d/da/337-3.png" width=95% height=70% style="display: block;margin: 10px auto;"/>
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<img src="https://static.igem.org/mediawiki/parts/d/d0/337-2.png" width=350 style=" margin: 10px auto;">  
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<img src="https://static.igem.org/mediawiki/parts/d/da/337-3.png" width=288 style=" margin: 10px auto;"/>
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<p style="text-align: center:">Figure 2. Schematic of gene circuits and kinetics of tetR inhibition in the Cell-Free system.<br>
 
<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>
 
(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>  
 
(B) The gene circuits contains an additional part P70-ClpXP that makes the tetR repressor degrade, and degfp expresses.<br>  
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(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><br>
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(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>
 
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Latest revision as of 12:03, 21 October 2021


Tet Repressor

The TetR binds to the Tet Operator and by that causes knockdown auf gene expression. (BB-2, RFC12).



Improvement by iGEM ZJUT-China 2021

As the improvement of the 10_Heidelberg registered part (BBa_K337028: tet Repressor), we, ZJUT-China 2021 newly registered partBBa_K3885126. through adding a degradation tag ssrA to the 3' end of the tetR (Figure 1), endowing the tetR a novel function—degradation.

Figure 1. Schematic of hydrolysis by ClpXP protein.

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 .

Results

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.

TetR with ssrA can be degraded by ClpXP, so that the flourescence intensity increased as a result of the inhibition released from tet Repressor.
Therefore, we gave the tetR a new function and improved this part.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 10
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 652
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 10
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 10
  • 1000
    COMPATIBLE WITH RFC[1000]