Part:BBa_K3015011

Riboswitch Sensor cassette

This genetic device was used to proof the functionality of a Riboswitch-based diagnostic method.

Usage and Biology

This is the genetic device we realized in the lab with a transcriptional Theophylline-Riboswitch. T7-Polymerase BBa_K3015006 will be expressed in the presence of Theophylline. The expression of T7-Polymerase induces the strong expression of amilCP BBa_K592009, a Blue Chromoprotein, that acts as visual readout. The expression of amilCP is under the control of the T7-Promoter BBa_K3015012. This makes up our composite part BBa_K3015003. The designed and constructed system can easily be adapted for detection of various disease-causing agents by just changing the riboswitch-sequence according to the target.

We could show that the part BBa_K3015011 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 composite part BBa K3015011 consists of the following two constructs (see figure 1 and 2):

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.


Figure 2: T7 promoter construct BBa_K3015003

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.


Figure 3: Overnight Cultures after 15 hours uninduced (left) and induced with 4mM Theophylline (right)

This table shows that even low concentrations of Theophylline can induce our signal cascade, resulting in a clear visual readout which can be seen in Figure 4 and Figure 5. The results are taken from our 2nd experiment which can be found under "Results" on our Wikipage with more details.

Table 1: Visual readout of different Theophylline concentrations over time

Figure 4 shows the samples from experiment 2 after being induced with different Theophylline concentrations and incubated for 1 hour (first row) and 2 hours (second row). The first microtube in each row is our negative control without being induced with Theophylline, the second column each row is the culture induced with 0.001 mM Theophylline, the third microtube is induced with 0.01 mM, followed by 0.1 mM, 1 mM and 5 mM inducer concentration. (from low to high-comparable with the concentrations in table 1)


Figure 4: Experiment 2 - samples with different Theophylline concentrations after 1h (first microtube row) and 2h (second row) of incubation


Figure 5: Experiment 2 - samples with different Theophylline concentrations after 3h (first microtube row) and 4h (second row) of incubation

Figure 5 shows the samples from experiment 2 after being induced with different Theophylline concentrations and incubated for 3 hours (first row) and 4 hours (second row).
The first microtube in each row is our negative control without being induced with Theophylline. In both Figure 3 and 4 the color change can be visually seen as described in Table 4.
Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 2886
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2820
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 2886
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 2886
  
• 1000
  COMPATIBLE WITH RFC[1000]