Difference between revisions of "Part:BBa K2333437"
 
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This part contains the CueR repressor under the constitutive promoter <partinfo>J23102</partinfo> combined with pdt-A tagged RFP under the control of the PcopA promoter. Protein degradation tag A is the strongest of the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (<partinfo>Bba_K2333011</partinfo>). Therefore, this part is the fastest of 6 pdt's to reach the steady-state fluorescence value. This part is contained in a suite of parts that allowed William and Mary 2017 to demonstrate persistence of speed-change effects for protein outputs beyond simple reporter proteins, and provides an example of a practical application of their speed-control system to improve biosensor output speed.  
 
This part contains the CueR repressor under the constitutive promoter <partinfo>J23102</partinfo> combined with pdt-A tagged RFP under the control of the PcopA promoter. Protein degradation tag A is the strongest of the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (<partinfo>Bba_K2333011</partinfo>). Therefore, this part is the fastest of 6 pdt's to reach the steady-state fluorescence value. This part is contained in a suite of parts that allowed William and Mary 2017 to demonstrate persistence of speed-change effects for protein outputs beyond simple reporter proteins, and provides an example of a practical application of their speed-control system to improve biosensor output speed.  
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<html><img src="https://static.igem.org/mediawiki/2017/b/bc/T--William_and_Mary--circuit4.jpeg" width="600px"/></html>
  
 
===Characterization===
 
===Characterization===
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<html><img src="https://static.igem.org/mediawiki/2017/d/da/T--William_and_Mary--Copper_Normalized_V1.png" width="600px"/></html>
 
<html><img src="https://static.igem.org/mediawiki/2017/d/da/T--William_and_Mary--Copper_Normalized_V1.png" width="600px"/></html>
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<html><img src="https://static.igem.org/mediawiki/parts/b/b3/T--William_and_Mary--Copper_Timecourse.png" width="600px"/></html>
 
  
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Graph 1: Time course measurements were performed according to standard protocol, and copper sensor parts were transformed on 1C3 along with <partinfo>K2333434</partinfo> on 3K3, and induced with 500μM copper sulfate and .01mM IPTG. In addition to requiring less IPTG than W&M 2017's other parts, copper parts worked well at 500μM CuSO4 but experienced die offs at 1000μM. Fluorescence was normalized to steady state based upon when fluorescence no longer increased.
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W&M 2017 was able to see a significant, but somewhat noisy speed change. While it was clear that parts with increased degradation rate were reaching steady state faster, it was fairly difficult to distinguish them from one another. This was likely due to cell death and toxicity issues with higher concentrations of copper sulfate and time concerns preventing thorough more testing of parameters.
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<html><img src="https://static.igem.org/mediawiki/parts/b/b3/T--William_and_Mary--Copper_Timecourse.png" width="650px"/></html>
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Graph 2: Results for a plate reader functionality assay. Cells were grown for 4 hours and then induced with either 500µM or 1mM of CuSO4 (to induce copper sensing parts). After 2 hours, cells were induced further with .01mM IPTG (to induce pLac mf-Lon). Introduction of mf-Lon clearly decreased the fluorescence of the cells, and based upon the results from this assay, W&M 2017 decided to perform speed tests on these constructs with 500µM CuSO4 and .01mM IPTG.
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<html><img src="https://static.igem.org/mediawiki/parts/3/38/T--William_and_Mary--copper_MEFL_0.png" width="650px"/></html>
  
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Time course measurements were performed according to standard protocol, and copper sensor parts were transformed on 1C3 along with <partinfo>K2333434</partinfo> on 3K3, and induced with 500μM copper sulfate and .01mM IPTG. In addition to requiring less IPTG than W&M 2017's other parts, copper parts worked well at 500μM CuSO4 but experienced die offs at 1000μM. Fluorescence was normalized to steady state based upon when fluorescence no longer increased. As the no-Lon condition had not reached steady state when time course was ended, it was normalized to the final collected data point, which is likely close to the true steady state.  
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Graph 3: Measurements of absolute gene expression using copper sensor constructs. Data is shown for each construct until steady state is reached (this means at least two consecutive subsequent data points do not increase in fluorescence). Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.
  
  

Latest revision as of 00:17, 2 November 2017


Copper sensor with Protein Degradation Tag A

This part constitutively expresses the CueR repressor. This repressor then inhibits the expression of protein degradation tagged (pdt-A) RFP from the downstream PcopA promoter. In the presence of copper, the repression of CueR is inhibited leading to greater RFP expression. Characterized by UMaryland 2017, fluorescence of this construct has been shown to increase with increasing copper concentration in both a qualitative and quantitative manner.

Usage and Biology

This part contains the CueR repressor under the constitutive promoter BBa_J23102 combined with pdt-A tagged RFP under the control of the PcopA promoter. Protein degradation tag A is the strongest of the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (BBa_K2333011). Therefore, this part is the fastest of 6 pdt's to reach the steady-state fluorescence value. This part is contained in a suite of parts that allowed William and Mary 2017 to demonstrate persistence of speed-change effects for protein outputs beyond simple reporter proteins, and provides an example of a practical application of their speed-control system to improve biosensor output speed.

Characterization

W&M 2017 characterized this tag's degradation rate and speed change effects as part of their iGEM project. The graphs below show the speed data of this pdt tagged copper sensor part along with the data from the other tags in this series (BBa_K2333437-BBa_K2333442).

Graph 1: Time course measurements were performed according to standard protocol, and copper sensor parts were transformed on 1C3 along with BBa_K2333434 on 3K3, and induced with 500μM copper sulfate and .01mM IPTG. In addition to requiring less IPTG than W&M 2017's other parts, copper parts worked well at 500μM CuSO4 but experienced die offs at 1000μM. Fluorescence was normalized to steady state based upon when fluorescence no longer increased.


W&M 2017 was able to see a significant, but somewhat noisy speed change. While it was clear that parts with increased degradation rate were reaching steady state faster, it was fairly difficult to distinguish them from one another. This was likely due to cell death and toxicity issues with higher concentrations of copper sulfate and time concerns preventing thorough more testing of parameters.


Graph 2: Results for a plate reader functionality assay. Cells were grown for 4 hours and then induced with either 500µM or 1mM of CuSO4 (to induce copper sensing parts). After 2 hours, cells were induced further with .01mM IPTG (to induce pLac mf-Lon). Introduction of mf-Lon clearly decreased the fluorescence of the cells, and based upon the results from this assay, W&M 2017 decided to perform speed tests on these constructs with 500µM CuSO4 and .01mM IPTG.

Graph 3: Measurements of absolute gene expression using copper sensor constructs. Data is shown for each construct until steady state is reached (this means at least two consecutive subsequent data points do not increase in fluorescence). Geometric mean of 10,000 cells each of three biological replicates. Shaded region represents one geometric standard deviation above and below the mean.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1229
    Illegal AgeI site found at 1341
  • 1000
    COMPATIBLE WITH RFC[1000]