Part:BBa_K1074011
PsdpRI+RBS+SDP
We construct this gene circuit as our kill switch to kill the engineered Bacillus subtilis for the safety purpose.Killing is mediated by the exported toxic protein SdpC. Extracellular SdpC induces the synthesis of an immunity protein, SdpI, which protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein. The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SdpC. When SdpC toxins are sensed, they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SdpC will be produced. Trapped in this endless loop, the SdpC producing cells fails to cope with enormous toxin SdpC and doomed after eliminating their siblings. Eventually, the group of engineered Bacillus subtilis is destroyed instead of sporulating.
Usage and Biology
We construct this gene circuit as our kill switch to kill the engineered Bacillus subtilis for the safety purpose.Killing is mediated by the exported toxic protein SdpC. Extracellular SdpC induces the synthesis of an immunity protein, SdpI, which protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein. The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SdpC. When SdpC toxins are sensed, they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SdpC will be produced. Trapped in this endless loop, the SdpC producing cells fails to cope with enormous toxin SdpC and doomed after eliminating their siblings. Eventually, the group of engineered Bacillus subtilis is destroyed instead of sporulating.
We have built model to verify the validity of this part, and our experiment is still going on.
There are both positive and negative feedback loops in this process. On the one hand, SdpI is unable to sequestrate the autorepressor, SdpR, until it captures the toxin, SdpC. The accumulation of SdpC will thus facilitate SdpI to capture more SdpR and thereby relieve the repression of SdpR, stimulating the expression of itself. This is the positive feedback loop which leads to the increasing accumulation of SdpC and finally the death of the bacteria. On the other hand, the removal of SdpR also enhance the expression of SdpI and accelerate the sequestration of SdpC, which forms a negative feedback loop whose effects contradict the positive feedback loop. However, since the copy number of SdpC is much higher, it is believed that the positive loop is strong enough to outweigh the negative one, which guarantees this mechanism will finally leads to collapse instead of equilibrium.
The ODE model of singular cells
There is no denying fact that the essential goal of engineered bacterias who carry this so called “suicide” locus itself is to kill their siblings rather than themselves to ensure the survival of themselves. Surly they can kill their siblings, but can they finally eliminate themselves, as we expects? The trivial experiment protocol and huge uncertainty had put off our experiment, and as expected, we failed to achieve the construction of complete reporter system in our laboratory. Fortunately, we could resort to mathematical models to verify the validity of this locus theoretically. There are six independent variables in individual cells, and the theoretically if the initial conditions are fixed, all of them will be the univariate functions of time. The following table illustrates the mark and meaning of each variable.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 98
Illegal SpeI site found at 680
Illegal PstI site found at 2107 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 98
Illegal SpeI site found at 680
Illegal PstI site found at 2107 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 98
Illegal SpeI site found at 680
Illegal PstI site found at 2107 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 98
Illegal SpeI site found at 680
Illegal PstI site found at 2107
Illegal AgeI site found at 867 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 854
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