Difference between revisions of "Part:BBa K1017726"
 
(8 intermediate revisions by 4 users not shown)
Line 3: Line 3:
  
  
−
We design this part to change heme into phycocyanobilin(PCB). The following is the change pathway.
+
We design this part to change heme into phycocyanobilin(PCB). The following is the pathway.
 +
The part of the photoreceptor that responds to light, phycocyanobilin, is not naturally produced in E. coli. We therefore introduced two phycocyanobilin-biosynthesis genes (ho1 and pcyA) from Synechocystis that convert heme into phycocyanobilin.
  
−
https://static.igem.org/mediawiki/2013/9/9b/Nctu_formosa_PCBsynthe.jpg
+
[[File:Nctu_formosa_PCBsynthe.jpg|300px|center]]
  
  
−
Pcons is a constitutive promoter family member(from J23100 to J23119) which can be used to tune the expression level of express part and we choose J23101. B0030 and B0032 are strong ribosome binding sites(RBS), but they have different strength. pcyA and hol1 are two requisite genes which are required for the biosynthesis to change heme into PCB. hol1 will oxidizes the heme group then generate biliverdin IXalpha, and pcyA converts biliverdin IXalpha into phycocyanobilin(PCB).
+
In this part, Pcons is a constitutive promoter family member(from J23100 to J23119) which can be used to tune the expression level of express part. And what we choose is J23101. B0030 and B0032 are strong ribosome binding sites(RBS), but they have different strength. pcyA and hol1 are two requisite genes which are required for the biosynthesis to change heme into PCB. hol1 will oxidizes the heme group then generate biliverdin IXalpha, and pcyA converts biliverdin IXalpha into phycocyanobilin(PCB).
−
 
+
  
 +
===Red promoter===
 +
<p>In order to positively regulate gene expression with red light, we employed '''this light receptor biobrick (K1017301)''' to give E. coli light sensing ability, and Pred biobrick for E. coli to respond to red light, both depicted in the first figure below. Pred is repressed in the dark and activated by red light.</p>
 +
[[file:NCTU_result_Pred_biobrick.png|center|600px|Notice that Pred includes a 37<sup>o</sup>C RBS that is only activated above or at 37<sup>o</sup>C. ]]
 +
To test whether red light and Cph8 can regulate P<sub>red</sub> or not, we measured the florescence expression of E. coli that were exposed to red light under 37 °C. The figure shows that red light can in fact activate P<sub>red</sub>. Colony 5 displays a high normalized expression under red light, suggesting that the biobrick was successfully activated by red light. In other words, our Cph8 can work. On the other hand, colony 3 might be mutated, as it shows only little expression.
 +
[[File:600px-NCTU result Activation Efficiency of light regulated system.png|600px|center|]]
 +
<p>Positive control: Pcons+mGFP. Negative control:tet 30. Pred is activated by red
 +
light and shows strong GFP expression that is close to the expression of the positive
 +
control.</p>
 +
[[File:NCTU_result_Pred_fluo.png|center|600px]]
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 06:01, 30 September 2013

Pcons+B0030+pcya+B0032+ho1+B0030


We design this part to change heme into phycocyanobilin(PCB). The following is the pathway. The part of the photoreceptor that responds to light, phycocyanobilin, is not naturally produced in E. coli. We therefore introduced two phycocyanobilin-biosynthesis genes (ho1 and pcyA) from Synechocystis that convert heme into phycocyanobilin.

Nctu formosa PCBsynthe.jpg


In this part, Pcons is a constitutive promoter family member(from J23100 to J23119) which can be used to tune the expression level of express part. And what we choose is J23101. B0030 and B0032 are strong ribosome binding sites(RBS), but they have different strength. pcyA and hol1 are two requisite genes which are required for the biosynthesis to change heme into PCB. hol1 will oxidizes the heme group then generate biliverdin IXalpha, and pcyA converts biliverdin IXalpha into phycocyanobilin(PCB).

Red promoter

In order to positively regulate gene expression with red light, we employed this light receptor biobrick (K1017301) to give E. coli light sensing ability, and Pred biobrick for E. coli to respond to red light, both depicted in the first figure below. Pred is repressed in the dark and activated by red light.

Notice that Pred includes a 37oC RBS that is only activated above or at 37oC.

To test whether red light and Cph8 can regulate Pred or not, we measured the florescence expression of E. coli that were exposed to red light under 37 °C. The figure shows that red light can in fact activate Pred. Colony 5 displays a high normalized expression under red light, suggesting that the biobrick was successfully activated by red light. In other words, our Cph8 can work. On the other hand, colony 3 might be mutated, as it shows only little expression.

600px-NCTU result Activation Efficiency of light regulated system.png

Positive control: Pcons+mGFP. Negative control:tet 30. Pred is activated by red light and shows strong GFP expression that is close to the expression of the positive control.

NCTU result Pred fluo.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 833
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 386
  • 1000
    COMPATIBLE WITH RFC[1000]