Difference between revisions of "Part:BBa K3447007"
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<partinfo>BBa_K3447007 short</partinfo>
 
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P<sub>BAD</sub> promoter is an inducible promoter, P<sub>BAD</sub> contains sequences of PC. It was verified by iGEM 2017 Amazonas_Brazil team that the induction effect of arabinose could be realized by using only <i>araC</i> and P<sub>BAD</sub>.<br>
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T7 polymerase stage 2 with pMag.
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===Usage and Biology===
 
===Usage and Biology===
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In the arabinose operon, it is regulated by the AraC operon and activates the expression of downstream arabinose metabolism genes in the presence of arabinose inducers.<br>
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In our project, we divided T7 RNA polymerase into two parts, respectively called T7<sub>NT</sub> (<partinfo>BBa_K3447008</partinfo>) and T7<sub>CT</sub> (<partinfo>BBa_K3447007</partinfo>) and attached on the nMag and pMag. Under blue light irradiation, these two parts undergo specific blue light dependent heterodimerization and assemble into complete T7 RNA polymerase, which acts on PT7 promoter (<partinfo>BBa_K3447005</partinfo>) and activates downstream gene expression. And the process is reversible, that is, when blue light is stopped, the dimerized T7 RNA polymerase dedimers and loses its function.<br><br>
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The L-Arabinose operon is naturally present in <i>E. coli</i>.The regulatory protein AraC ACTS as a dimer. The operon contains two cis-type control elements: The P<sub>C</sub> promoter for the synthesis of AraC and the P<sub>BAD</sub> for the synthesis of the enzyme needed for the catabolism of L-Arabinose. P<sub>BAD</sub> contains three half sites, each of which binds to a subunit of ARAC-O2, I1, and I2. The O1 site is composed of O1L and O1R half sites that bind two subunits. Half of O2 is in the araC coding region.
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We connected the target genes downstream of P<sub>BAD</sub> to constitute the effect part of arabinose sensing system, and constructed the whole arabinose sensing system on the same plasmid with P<sub>C</sub> and araC.  
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Arabinose induces endotoxin expression: <partinfo>BBa_K3447000</partinfo>
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Arabinose induces green fluorescent protein expression: (链接a01+c09)
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[[Image: T7 promoter (strong promoter from T7 bacteriophage).jpg|center|frame|100px|<b>Figure 1. </b> Main principles of the molecular mechanism of T7 promoter]]<br><br>
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In our project, we use this part to form a complete T7 RNA polymerase by dimerizing it with T7<sub>NT</sub> under blue light. (链接104+f09+104+f10+001+b17)<br>
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3447007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3447007 SequenceAndFeatures</partinfo>
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==<b>Design</b>==
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===Design Notes===
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We added some synonymous mutations to avoid part rules.
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*Armin Baumschlager, Stephanie K. Aoki, and Mustafa Khammash, Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control, <i>ACS Synth. Biol.</i> 2017, <b>6</b>, 2157-2167.<br><br>
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===References===
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1. Armin Baumschlager, Stephanie K. Aoki, and Mustafa Khammash, Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control, <i>ACS Synth. Biol.</i> 2017, <b>6</b>, 2157-2167.<br><br>
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Revision as of 09:52, 25 October 2020


xynD, which produces xylanase

T7 polymerase stage 2 with pMag.


Usage and Biology

In our project, we divided T7 RNA polymerase into two parts, respectively called T7NT (BBa_K3447008) and T7CT (BBa_K3447007) and attached on the nMag and pMag. Under blue light irradiation, these two parts undergo specific blue light dependent heterodimerization and assemble into complete T7 RNA polymerase, which acts on PT7 promoter (BBa_K3447005) and activates downstream gene expression. And the process is reversible, that is, when blue light is stopped, the dimerized T7 RNA polymerase dedimers and loses its function.

Figure 1. Main principles of the molecular mechanism of T7 promoter


In our project, we use this part to form a complete T7 RNA polymerase by dimerizing it with T7NT under blue light. (链接104+f09+104+f10+001+b17)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 216
    Illegal PstI site found at 226
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 216
    Illegal PstI site found at 226
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 216
    Illegal PstI site found at 226
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 216
    Illegal PstI site found at 226
  • 1000
    COMPATIBLE WITH RFC[1000]

Design

Design Notes

We added some synonymous mutations to avoid part rules.



  • Armin Baumschlager, Stephanie K. Aoki, and Mustafa Khammash, Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control, ACS Synth. Biol. 2017, 6, 2157-2167.

References

1. Armin Baumschlager, Stephanie K. Aoki, and Mustafa Khammash, Dynamic Blue Light-Inducible T7 RNA Polymerases (Opto-T7RNAPs) for Precise Spatiotemporal Gene Expression Control, ACS Synth. Biol. 2017, 6, 2157-2167.