Difference between revisions of "Part:BBa K3015003"

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<partinfo>BBa_K3015003 short</partinfo>
 
<partinfo>BBa_K3015003 short</partinfo>
  
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This composite Part expresses the blue chromoprotein amilCP [[Part:BBa_K592009|BBa_K592009]] in the presence of T7-Polymerase.
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This composite Part expresses the blue chromoprotein amilCP [[Part:BBa_K592009|BBa_K592009]] in the presence of T7-Polymerase [[Part:BBa_K3015006|BBa_K3015006]] under the control of T7-Promoter [[Part:BBa_K3015012|BBa_K3015012]].
  
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===Usage and Biology===
 
===Usage and Biology===
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An important aspect of our iGEM BOKU-Vienna Team’s new diagnostic method for Buruli Ulcer is the simple and clear visual readout that does not require complex equipment or training. For that the composite part BBa_K3015003 played an important role, as a visual readout signal at the end of a signal-cascade that will be initially induced by Theophylline [[Part:BBa_K3015011|BBa_K3015011]]. In the presence of Theophylline the T7-Polymerase [[Part:BBa_K3015006|BBa_K3015006]] can be produced and will then activate the production of amilCP [[Part:BBa_K592009|BBa_K592009]] under the control of the T7-Promoter [[Part:BBa_K3015012|BBa_K3015012]].
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<br>
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We could show that the part BBa_K3015003 works very well based on the positive signal due to the binding of an inducer to the aptamer, which can be easily seen with the naked eye. Our team took pictures of spun-down microtubes consisting of Escherichia coli (DH10B) strain induced under different concentrations of Theophylline and different incubation time. <br>
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<br>
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The tested expression cassette consists of the following two constructs (see figure 1 and 2):<br>
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[[File:T--BOKU-Vienna--T7-Polymerase_Construct.png|400px]]<br>
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Figure 1: T7 Polymerase construct<br>
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<br>
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Figure 1 shows our T7-Polymerase under the control of a Theophylline inducible riboswitch. If T7-Polymerase is beeing expressed in the cell it will bind to the T7-Promoter and expression of amilCP (see figure 2) takes place. The amount of expressed amilCP is proportional to the presence of T7-polymerase.<br>
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<br>
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[[File:T--BOKU-Vienna--T7-Promoter_Construct.png|400px]]<br>
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Figure 2: T7 promoter construct<br>
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<br>
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For proving the functioning of the switch, we set up overnight cultures with the plasmid construct and induced them with different Theophylline concentrations. In addition to that, negative samples with the bacteria including our construct with no concentration of the toxin were added for the ability to check the leakiness of our promoter. In our pre-experiment two separate overnight cultures were incubated without Theophylline (left) and with 4mM Theophylline (right) see figure 3.<br>
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<br>
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[[File:T--BOKU-Vienna--BB3_overnight_test.png|400px]]<br>
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Figure 3: Overnight Cultures after 15 hours uninduced (left) and induced with 4mM Theophylline (right)<br>
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Latest revision as of 23:22, 21 October 2019


T7prom-amilCP-Term

This composite Part expresses the blue chromoprotein amilCP BBa_K592009 in the presence of T7-Polymerase BBa_K3015006 under the control of T7-Promoter BBa_K3015012.

Usage and Biology

An important aspect of our iGEM BOKU-Vienna Team’s new diagnostic method for Buruli Ulcer is the simple and clear visual readout that does not require complex equipment or training. For that the composite part BBa_K3015003 played an important role, as a visual readout signal at the end of a signal-cascade that will be initially induced by Theophylline BBa_K3015011. In the presence of Theophylline the T7-Polymerase BBa_K3015006 can be produced and will then activate the production of amilCP BBa_K592009 under the control of the T7-Promoter BBa_K3015012.


We could show that the part BBa_K3015003 works very well based on the positive signal due to the binding of an inducer to the aptamer, which can be easily seen with the naked eye. Our team took pictures of spun-down microtubes consisting of Escherichia coli (DH10B) strain induced under different concentrations of Theophylline and different incubation time.

The tested expression cassette consists of the following two constructs (see figure 1 and 2):
T--BOKU-Vienna--T7-Polymerase Construct.png
Figure 1: T7 Polymerase construct

Figure 1 shows our T7-Polymerase under the control of a Theophylline inducible riboswitch. If T7-Polymerase is beeing expressed in the cell it will bind to the T7-Promoter and expression of amilCP (see figure 2) takes place. The amount of expressed amilCP is proportional to the presence of T7-polymerase.

T--BOKU-Vienna--T7-Promoter Construct.png
Figure 2: T7 promoter construct


For proving the functioning of the switch, we set up overnight cultures with the plasmid construct and induced them with different Theophylline concentrations. In addition to that, negative samples with the bacteria including our construct with no concentration of the toxin were added for the ability to check the leakiness of our promoter. In our pre-experiment two separate overnight cultures were incubated without Theophylline (left) and with 4mM Theophylline (right) see figure 3.

T--BOKU-Vienna--BB3 overnight test.png
Figure 3: Overnight Cultures after 15 hours uninduced (left) and induced with 4mM Theophylline (right)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 73
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 7
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 73
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 73
  • 1000
    COMPATIBLE WITH RFC[1000]