Difference between revisions of "Part:BBa K606036"
 
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<partinfo>BBa_K606036 short</partinfo>
 
<partinfo>BBa_K606036 short</partinfo>
  
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T7 RNA polymerase auto-amplifier system for B. Subtilis and E. coli
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T7 RNA polymerase auto-amplifier system (''T7 autoloop'') for B. Subtilis and E. coli
  
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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Many systems in biology are very tedious. Though, they are difficult to observe directly or by contructing reporter genes with promoters.  
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This construct, the so-called T7 autoloop, uses positive feedback to create signal amplification.
  
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If you want to detect the presence of a tiny phenomena, the synthetic biologist has to rely on a signal amplifier, to detect it's signal. The problem of building a positive feed-back loop auto-amplifier is the sensitivity to noise. The standard proteins that binds to DNA (LacI, TetR...) present a too important leakage for controling such a design.
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The T7 RNA polymerase has a very specific promoter (pT7, 25 nucleotides) that can be recognize by no other polymerases. The system is really orthogonal as long as there is no polymerase leakeage from the transcription in the plasmid. Synthetic biology plasmids (here pSB1C3) use 4 standards terminators before the promoter to limit such leakage. You might need to add some others if you are working on another plasmid.
  
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The T7 RNA polymerase has a very specific promoter, of 25 nucleotides, that can be recognize by no other polymerases. Though, the system is really orthogonal, as long as there is no polymerase leakeage from the transcription in the plasmid. Synthetic biology plasmids (here pSB1C3) are protected from plasmid noise by 4 standards terminators. You might need to add some others if you are working on a non silent plasmid.
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The stochastic leakeage of this system has been characterized (see the experiments page). Once the system is activated, the cell stop dividing and glows very high.
  
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The leakeage of this system has been characterized (see the experiments page), and it is shown that there is very little false positive. Once the system is activated, the cell stop deviding and glow very high.
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You can use this part directly to detect a tiny amount of T7 RNA polymerase in a cell (in the case of a cell infection for instance).
  
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You can use this part directly to detect a tiny amount of T7 RNA polymerase in a cell (in the case of a cell infection for instance), or by cloning the promoter you want to test in front of this construct.
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The RBS used here are for B. subtilis but the system works fine in E. coli as well (as shown in the experiments).
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The RBS used here are for B. subtilis but the system works fine in E. coli as well.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 12:25, 21 October 2011

pT7 SpoVG T7 SpoVG GFP T7terminator.

T7 RNA polymerase auto-amplifier system (T7 autoloop) for B. Subtilis and E. coli


Usage and Biology

This construct, the so-called T7 autoloop, uses positive feedback to create signal amplification.

The T7 RNA polymerase has a very specific promoter (pT7, 25 nucleotides) that can be recognize by no other polymerases. The system is really orthogonal as long as there is no polymerase leakeage from the transcription in the plasmid. Synthetic biology plasmids (here pSB1C3) use 4 standards terminators before the promoter to limit such leakage. You might need to add some others if you are working on another plasmid.

The stochastic leakeage of this system has been characterized (see the experiments page). Once the system is activated, the cell stop dividing and glows very high.

You can use this part directly to detect a tiny amount of T7 RNA polymerase in a cell (in the case of a cell infection for instance).

The RBS used here are for B. subtilis but the system works fine in E. coli as well (as shown in the experiments).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3598
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3374