Difference between revisions of "Part:BBa K1031021"
Line 9: Line 9:
 
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.   
 
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.   
  
−
https://static.igem.org/mediawiki/igem.org/4/4b/Peking2013_model9.png
+
<html>
 +
<img src="https://static.igem.org/mediawiki/igem.org/4/4b/Peking2013_model9.png", width=500px;/>
 +
</html>
 +
 
 +
'''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.
  
 
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).  
 
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).  
Line 23: Line 27:
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
  
 
<!-- -->
 
<!-- -->

Revision as of 07:45, 25 September 2013

Hybrid Promoter for Band-pass Filter

hybrid promoter for the band-pass filter


Construction of our hybrid promoter

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.

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.

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).

Peking2013_Construction_of_hybrid_promoter.png

Figure 2.The construction of our hybrid promoter.

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.


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