Difference between revisions of "Part:BBa K118011:Experience"

(→‎User Reviews)
(→‎User Reviews)
Line 48: Line 48:
 
<I>WHU-China 2012</I>
 
<I>WHU-China 2012</I>
 
|width='60%' valign='top'|
 
|width='60%' valign='top'|
−
Firstly,by embedding the gene of RFP downstream the promoter PcstA,we have successfully certified that the promoter PcstA can be activated by CRP.In other words, it can be repressed by high concentration of glucose.
 
−
What's more, we have constructed a indirect pathway by using an intermediate to change the function of the promoter. In the indirect pathway,PcstA is activated by high concentration of glucose.
 
  
 +
Firstly,by embedding the gene of RFP downstream the promoter PcstA,we have successfully certified that the promoter PcstA can be activated by CRP.In other words, it can be repressed by high concentration of glucose.
 +
What's more, we have constructed an indirect pathway by using an intermediate to change the function of the promoter. In the indirect pathway,PcstA is activated by high concentration of glucose.
 +
<html>
 +
<p align="center"><img src="https://static.igem.org/mediawiki/2012/d/d5/Indirect_regulation.png" width="500"
 +
height="250" hspace="2" vspace="1" align="middle" /></p>
 +
<p align="center"> The indirect regulatory pathway</p>
 
After construction,the correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader.All fluorescence was normalized with absorbance at 600 nm.The cell density was characterized by the absorbance at 600nm,which increased with glucose concentration.The result is showed in the figure bellow.   
 
After construction,the correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader.All fluorescence was normalized with absorbance at 600 nm.The cell density was characterized by the absorbance at 600nm,which increased with glucose concentration.The result is showed in the figure bellow.   
  
−
<html>
+
 
 
<p align="center"><img name="" src="https://static.igem.org/mediawiki/2012/d/d5/Fluorescence_1n.png" width="520" height="409" alt=""></p>
 
<p align="center"><img name="" src="https://static.igem.org/mediawiki/2012/d/d5/Fluorescence_1n.png" width="520" height="409" alt=""></p>
 
<p algin="justify">
 
<p algin="justify">

Revision as of 17:57, 26 October 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K118011

Preliminary tests of this part were conducted using the reporter gene xylE (BBa_K118021). Strong repression occurred in the presence of glucose, and partial repression in the presence of high concentrations of higher sugars. Results can be found [http://2008.igem.org/Team:Edinbrugh/Results/PcstA-xylE here].

Characterization of PcstA with RFP Generator Part:BBa K081014

User Reviews

UNIQ164136b71c075886-partinfo-00000001-QINU

BBa_K118011 1 Not understood Kun


II09 CRP-GFP fluor different media.jpg

Cells with BBa_K200018 were grown overnight in various different media, and the GFP fluorescence was measured.

After overnight culture, the corrected fluorescence of glucose is almost negligible, showing that glucose represses the PcstA promoter strongly.

For all the other secondary carbon sources, 10% Casamino Acids in M9 shows the highest corrected fluorescence at 22000.

For more information, go to the Imperial iGEM 2009 E.ncapsulator project page on [http://2009.igem.org/Team:Imperial_College_London/Wetlab/Results/CRP_and_Media BBa_K200018 testing results]

;

UNIQ164136b71c075886-partinfo-00000003-QINU

UNIQ164136b71c075886-partinfo-00000004-QINU

BBa_K118011 2 Not understood WHU-China 2012

Firstly,by embedding the gene of RFP downstream the promoter PcstA,we have successfully certified that the promoter PcstA can be activated by CRP.In other words, it can be repressed by high concentration of glucose. What's more, we have constructed an indirect pathway by using an intermediate to change the function of the promoter. In the indirect pathway,PcstA is activated by high concentration of glucose.

The indirect regulatory pathway

After construction,the correct clones were cultured in 96-well plate at 37℃ for 24 hours,then the fluorescence and absorbance at 600 nm were recorded on a SpectraMax M2 plate reader.All fluorescence was normalized with absorbance at 600 nm.The cell density was characterized by the absorbance at 600nm,which increased with glucose concentration.The result is showed in the figure bellow.

In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration. In another word, the indirect pathway can be activated by glucose

For more information, please go to wiki of WHU-China 2012 on [http://2012.igem.org/Team:WHU-China/Project?catalog=2#Indirect_Pathway_Design Indirect pathway]

;

UNIQ164136b71c075886-partinfo-00000007-QINU