Difference between revisions of "Part:BBa K2150022"
 
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===Usage and Biology===
 
===Usage and Biology===
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We design this part to test whether the introduction of T7 polymerase and T7 promoter has the potential to enhance our scavenger's ability to degrade tetracycline in presence of tetR. In this part, pure GFP is produced, so that the less toxic tetracycline analog anhydrotetracycline (aTc) is not degraded. And then our design can be tested under a constant concentration of Tc (represented by the concentration of aTc). We call this a simulation system.
+
We design this part to test whether the introduction of T7 polymerase and T7 promoter has the potential to enhance our scavenger's ability to degrade tetracycline in the presence of tetR. In this part, pure GFP is produced, so that the less toxic tetracycline analog anhydrotetracycline (aTc) is not degraded. And then our design can be tested under a constant concentration of Tc (represented by the concentration of aTc). We call this a simulation system.
  
 
===Characterization===
 
===Characterization===
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The expression level of GFP rises as the concentration of aTc increases(Fig.1), in the presence of tetR, which indicates the DNA downstream of T7 promoter is inducible by tetracycline and its analogs.  
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The expression level of GFP rose as the concentration of aTc increased(Fig.1) in the presence of tetR, which indicated that the DNA downstream of T7 promoter was inducible by tetracycline and its analogs.  
  
  
 
                                 [[File:Captain Simulator_1.png|250px]]
 
                                 [[File:Captain Simulator_1.png|250px]]
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Comparing to another part (see [https://parts.igem.org/Part:BBa_K2150014 BBa_K2150014]), in the presence of tetR, this part performs better (emits a higher level of fluorescence) when the concentratiion of atc is low(Fig.2a, Fig.2b).
+
Comparing to another part (see [https://parts.igem.org/Part:BBa_K2150014 BBa_K2150014]), we found that in the presence of tetR, this part performed better (emitted a higher level of fluorescence) when the concentratiion of aTc was low(Fig.2a, Fig.2b).
  
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                                [[File:Captain Simulator_2.png|250px]]
+
              [[File:Captain Simulator_2.png|500px]]
  
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However when the concentration of aTc is high, this part will inhibit the cell growth(fig.3a), which leads to a weaker fluorescence intensity than BBa_2150014(Fig.3b). The reason for this is that when pTet is fully induced, T7 RNA polymerase is produced more than enough. Such a large quantity of T7 RNAPs caused a short of resources such as nucleotides in the cell, which inhibited the synthesis of other proteins and eventually led to the inhibition of cell growth. Operating this part on a low or medium copy plasmid may solve this probelm.
+
However when the concentration of aTc was high, this part inhibited the cell growth(fig.3a), which led to a lower fluorescence intensity than BBa_2150014(Fig.3b). The reason for this is that when pTet was fully induced, T7 RNA polymerase was produced more than enough. Such a large quantity of T7 RNAPs caused a short of resources such as nucleotides in the cell, which inhibited the synthesis of other proteins and eventually led to the inhibition of cell growth. Operating this part on a low or medium copy plasmid may solve this probelm.
  
−
                                [[File:Captain Simulator_3.png|250px]]
+
              [[File:Captain Simulator_3.png|500px]]
  
 
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Latest revision as of 01:18, 20 October 2016


pTet+RBS+T7RNAP+Ter+pT7+RBS+GFP+DT

In this part, T7 RNA polymerase(T7RNAP) is expressed from pTet which is inducible by tetracycline and its analogs. T7RNAP can activate the T7 promoter, thus producing GFP in large quantity.


Usage and Biology

We design this part to test whether the introduction of T7 polymerase and T7 promoter has the potential to enhance our scavenger's ability to degrade tetracycline in the presence of tetR. In this part, pure GFP is produced, so that the less toxic tetracycline analog anhydrotetracycline (aTc) is not degraded. And then our design can be tested under a constant concentration of Tc (represented by the concentration of aTc). We call this a simulation system.

Characterization

The expression level of GFP rose as the concentration of aTc increased(Fig.1) in the presence of tetR, which indicated that the DNA downstream of T7 promoter was inducible by tetracycline and its analogs. 


                                Captain Simulator 1.png

Comparing to another part (see BBa_K2150014), we found that in the presence of tetR, this part performed better (emitted a higher level of fluorescence) when the concentratiion of aTc was low(Fig.2a, Fig.2b). 

              Captain Simulator 2.png

However when the concentration of aTc was high, this part inhibited the cell growth(fig.3a), which led to a lower fluorescence intensity than BBa_2150014(Fig.3b). The reason for this is that when pTet was fully induced, T7 RNA polymerase was produced more than enough. Such a large quantity of T7 RNAPs caused a short of resources such as nucleotides in the cell, which inhibited the synthesis of other proteins and eventually led to the inhibition of cell growth. Operating this part on a low or medium copy plasmid may solve this probelm. 

              Captain Simulator 3.png

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.rc site found at 3488