Difference between revisions of "Part:BBa K2384014"
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| + | __NOTOC__ | ||
| + | <partinfo>BBa_K23840014 short</partinfo> | ||
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| + | ===Usage and Biology=== | ||
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In our project, to increase the robustness of these biocontainment states, the circuit uses a genetic ‘toggle switch’ architecture in which reciprocal repression by the LacI and TetR transcription factors form transcription states that are maintained by the circuit’s linked feedback loops. | In our project, to increase the robustness of these biocontainment states, the circuit uses a genetic ‘toggle switch’ architecture in which reciprocal repression by the LacI and TetR transcription factors form transcription states that are maintained by the circuit’s linked feedback loops. | ||
To create a circuit in which the death state is dominant in the absence of the survival signal, the ribosome binding site (RBS) strengths of LacI and TetR to favor TetR expression was altered in a single-copy plasmid. In the resulting monostable circuit, the presence of the TetR inhibitor anhydrotetracycline (ATc) is required to maintain the circuit in the subordinate LacI+ survival state. Incorporation of toxin genes into the TetR+ state creates a kill switch where the presence of ATc is required to block toxin expression and cell death. | To create a circuit in which the death state is dominant in the absence of the survival signal, the ribosome binding site (RBS) strengths of LacI and TetR to favor TetR expression was altered in a single-copy plasmid. In the resulting monostable circuit, the presence of the TetR inhibitor anhydrotetracycline (ATc) is required to maintain the circuit in the subordinate LacI+ survival state. Incorporation of toxin genes into the TetR+ state creates a kill switch where the presence of ATc is required to block toxin expression and cell death. | ||
The circuit included additional palindromic LacI operator sites in the toxin gene promoter to minimize leaky toxin expression and introduced a transcriptional terminator upstream of the promoter to insulate the gene from spurious transcription. To accelerate the circuit’s switching dynamics, a degradation tag that is specifically recognized by mf-Lon20, a heterologous protease under control of a LacI-dependent promoter was fused to the C terminus of LacI. Upon removal of ATc, TetR repression of LacI allows expression of mf-Lon, which targets LacI for degradation to create a positive feedback loop that accelerates the switch to the TetR+ state. | The circuit included additional palindromic LacI operator sites in the toxin gene promoter to minimize leaky toxin expression and introduced a transcriptional terminator upstream of the promoter to insulate the gene from spurious transcription. To accelerate the circuit’s switching dynamics, a degradation tag that is specifically recognized by mf-Lon20, a heterologous protease under control of a LacI-dependent promoter was fused to the C terminus of LacI. Upon removal of ATc, TetR repression of LacI allows expression of mf-Lon, which targets LacI for degradation to create a positive feedback loop that accelerates the switch to the TetR+ state. | ||
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| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K2384000 SequenceAndFeatures</partinfo> | ||
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
| + | ===Functional Parameters=== | ||
| + | <partinfo>BBa_K2384000 parameters</partinfo> | ||
| + | <!-- --> | ||
Revision as of 07:23, 31 October 2017
No part name specified with partinfo tag.
Usage and Biology
In our project, to increase the robustness of these biocontainment states, the circuit uses a genetic ‘toggle switch’ architecture in which reciprocal repression by the LacI and TetR transcription factors form transcription states that are maintained by the circuit’s linked feedback loops. To create a circuit in which the death state is dominant in the absence of the survival signal, the ribosome binding site (RBS) strengths of LacI and TetR to favor TetR expression was altered in a single-copy plasmid. In the resulting monostable circuit, the presence of the TetR inhibitor anhydrotetracycline (ATc) is required to maintain the circuit in the subordinate LacI+ survival state. Incorporation of toxin genes into the TetR+ state creates a kill switch where the presence of ATc is required to block toxin expression and cell death. The circuit included additional palindromic LacI operator sites in the toxin gene promoter to minimize leaky toxin expression and introduced a transcriptional terminator upstream of the promoter to insulate the gene from spurious transcription. To accelerate the circuit’s switching dynamics, a degradation tag that is specifically recognized by mf-Lon20, a heterologous protease under control of a LacI-dependent promoter was fused to the C terminus of LacI. Upon removal of ATc, TetR repression of LacI allows expression of mf-Lon, which targets LacI for degradation to create a positive feedback loop that accelerates the switch to the TetR+ state.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]