Difference between revisions of "Part:BBa K1031021"

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== Construction of our hybrid promoter ==
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== '''Construction of our hybrid promoter''' ==
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The hybrid promoter is constructed for the band-pass filter. A band-pass filter can be theoretically summarized as an incoherent feed forward loop, consisted of a positive loop and a negative loop. '''(Fig. 1)'''As for the reporter node, a special promoter is used so that the reporter can be controlled by the activator and inhibitor both. 
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Band-pass filter constructed by Peking iGEM 2013 team: http://2013.igem.org/Team:Peking/Project/BandpassFilter
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<img src="https://static.igem.org/mediawiki/igem.org/4/4b/Peking2013_model9.png", width=500px;/>
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We modified a bacteriophage ϕR73’s <em>P<sub>2</sub></em> promoter into a hybrid promoter that can be activated by the ϕR73&delta; activator and repressed by the repressor cI simultaneously and put reporter sfGFP under its regulation. We constructed the hybrid promoter by replacing the sequence between position -1 and -25  of <em>P<sub>2</sub></em> promoter with the cI binding site <em>OR1</em> from Phage &lambda; <em>P<sub>R</sub></em> promoter. When ϕR73&delta; activator binds to its target sequence upstream of -35 element of the hybrid promoter, the transcription will start. The binding of cI dimers downstream of -35 element will block the binding of &sigma;<sup>70</sup> factors and thus repress the transcription even when ϕR73&delta; is bound. (<B>Fig. 1</B>). </p>
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'''Fig.1''': The basic topology of band-pass filter, a positive loop and a negative loop is shown. The yellow arrow indicates activation and black line indicates inhibition. A and B nodes are determined by parameter sensitivity analysis. this part represents the reporter, shown in the bottom of the picture.
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After deciding the circuit and protein to use, we started to design the promoter that could be activated by phiR73δ and be repressed by cI. To obtain a promoter, a hybrid promoter is required for the reporter node. We modified bacteriophage φR73’s promoter into a hybrid promoter that can be activated by the PhiR73 delta activator and repressed by the repressor cI respectively. We replaced the sequence between the -10 and -35 elements with the cI binding site Or1 from Phage λ. PhiR73δ activator binds to its binding site and the transcription starts. The binding of cI dimers will block the binding of σ70 factors and therefore repress the transcription (Fig.2).  
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https://static.igem.org/mediawiki/2013/4/4f/Peking2013_Construction_of_hybrid_promoter.png
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'''Figure 2'''.The construction of our hybrid promoter.
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Based on this construction, when the input signal is weak, the concentration of PhiR73 delta is too low to generate a strong output. When the input signal is medium, despite a portion of promoters occupied by cI dimmers, the rest still can be activated by PhiR73 delta and bring about a visible output. When the input signal is strong, almost all of the promoters are “conquered” by cI dimers. The transcription is blocked and the output is shut down. Hence only medium input signal can generate a significant output and the Band Pass Filter was made.
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<img src="https://static.igem.org/mediawiki/2013/b/bf/Peking2013_hybrid_promoter_2.2.png" style="width:800px;margin-left:60px" ></a>
  
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<B>Figure.1</B> Construction of Our Hybrid Promoter.</br> Sequence information of phage &phi;R73 <em>P<sub>2</sub></em> promoter (<b>a</b>), phage &lambda; <em>P<sub>R</sub></em> promoter (<b>b</b>) and our hybrid promoter (<b>c</b>) are shown. <b>a</b>, In the <em>P<sub>2</sub></em> promoter, &phi;R73&delta; binds to a region between position -42 and -71 and activates transcription. <b>b</b>, In <em>P<sub>R</sub></em> promoter, cI dimer binds to <em>OR1</em> site (marked as blue) between position -9 and -25, blocking binding of  &sigma;<sup>70</sup> factors and inhibiting transcription. cI binding region indicates the sequence we used to replace the corresponding region in <em>P<sub>2</sub></em> promoter.<b>c</b>, The hybrid promoter is constructed by replacing sequence between position -1 and -25 of &phi;R73 <em>P<sub>2</sub></em> promoter with sequence at the same position in phage &lambda; <em>P<sub>R</sub></em> promoter that contains an <em>OR1</em> site. The hybrid promoter is co-regulated by &phi;R73&delta; and cI, with &phi;R73&delta; activating and cI repressing. The repression of cI dominates over the activation of  &phi;R73&delta;, since the steric hindrance created by cI dimer prevents formation of  transcription initiation complex even when RNA polymerases are recruited through the help of  &phi;R73&delta;.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 18:48, 27 September 2013

Hybrid Promoter for Band-pass Filter

hybrid promoter for the band-pass filter


Construction of our hybrid promoter

We modified a bacteriophage ϕR73’s P2 promoter into a hybrid promoter that can be activated by the ϕR73δ activator and repressed by the repressor cI simultaneously and put reporter sfGFP under its regulation. We constructed the hybrid promoter by replacing the sequence between position -1 and -25 of P2 promoter with the cI binding site OR1 from Phage λ PR promoter. When ϕR73δ activator binds to its target sequence upstream of -35 element of the hybrid promoter, the transcription will start. The binding of cI dimers downstream of -35 element will block the binding of σ70 factors and thus repress the transcription even when ϕR73δ is bound. (Fig. 1).

Figure.1 Construction of Our Hybrid Promoter.
Sequence information of phage φR73 P2 promoter (a), phage λ PR promoter (b) and our hybrid promoter (c) are shown. a, In the P2 promoter, φR73δ binds to a region between position -42 and -71 and activates transcription. b, In PR promoter, cI dimer binds to OR1 site (marked as blue) between position -9 and -25, blocking binding of σ70 factors and inhibiting transcription. cI binding region indicates the sequence we used to replace the corresponding region in P2 promoter.c, The hybrid promoter is constructed by replacing sequence between position -1 and -25 of φR73 P2 promoter with sequence at the same position in phage λ PR promoter that contains an OR1 site. The hybrid promoter is co-regulated by φR73δ and cI, with φR73δ activating and cI repressing. The repression of cI dominates over the activation of φR73δ, since the steric hindrance created by cI dimer prevents formation of transcription initiation complex even when RNA polymerases are recruited through the help of φR73δ.

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 AgeI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 127