Part:BBa_K3015003
T7prom-amilCP-Term
This composite Part expresses the blue chromoprotein amilCP BBa_K592009 in the presence of T7-Polymerase.
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. We developed a riboswitch-based readout that can be interpreted with the naked eye. The riboswitch consists of an aptamer- based induction of T7- Polymerase (BBa_K3015006) and amilCP BBa_K592009 under the control of the T7- Promoter (BBa_K3015012).
The qualitative measurements are based on the expression of blue chromoprotein (amilCP) for a 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 spinned-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):
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
First, we wanted to make sure that the binding of Theophylline to the aptamer sequence would induce a chain of reactions starting with a confirmation change by which the aptamer loses its hairpin structure. Due to this the ribosome can transcribe the ribosome binding site and translation of the T7- Polymerase takes place. T7- Polymerase binds to the T7- Promoter which results in the expression of amilCP allowing a clear visual readout due to the color change.
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)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 73
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 7
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 73
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 73
- 1000COMPATIBLE WITH RFC[1000]
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