Difference between revisions of "Part:BBa K861169:Experience"

 
(→‎Applications of BBa_K861169)
 
(5 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
Line 5: Line 4:
  
 
===Applications of BBa_K861169===
 
===Applications of BBa_K861169===
 +
 +
<html>
 +
<h2>Device Design</h2>
 +
<p algin="justify">
 +
In this design, we used promoter PcstA and an intermediate regulator cI, as PcstA is activated by CRP in low glucose concentration, whereas we need a device boosted by high concentration of glucose.
 +
As you can see, in this pathway, protein cI will be expressed when CRP is activated under low glucose concentration.The large amount of cI repressed the promoter R0051 heavily. On the contrary, when glucose concentration is high, the promoter is derepressed.</p>
 +
<p algin="justify">
 +
We made RFP as reporter and certify that the design works well via two different methods.</p>
 +
 +
<h2>Fluorescence measurement</h2>
 +
<p algin="justify">
 +
In the first method, Fluorescence intensity of cell culture was directly read from a plate reader. And all data is normalized by OD600.
 +
</p>
 +
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/9/9c/Indirect_Fig_1.png" width="506" height="409" alt=""></p>
 +
 +
<p algin="justify">
 +
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, indicating that the device works as expected.</p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/d/dc/Indirect_Fig_2.png" width="500" height="220" alt=""></p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/2/21/Indirect_Fig_3.png" width="506" height="189" alt=""></p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/c/c3/Indirect_Fig_4.png" width="500" height="226" alt=""></p>
 +
<p algin="justify">
 +
The second method is practiced for single cells. The fluorescence pictures of different glucose concentration were captured by fluorescence microscope. Also, a program named <a href="http://2012.igem.org/Team:WHU-China/Modeling?catalog=3">FANCY</a> was designed to recognize single cell and calculate the fluorescence strength in the images. As the result shows, it conformed well to that of the first method.</p>
 +
 +
<p algin="justify">
 +
All these results indicated that the device we designed can meet the demand, promoters activated by glucose.
 +
</p>
 +
</html>
  
 
===User Reviews===
 
===User Reviews===
Line 17: Line 44:
 
Enter the review inofrmation here.
 
Enter the review inofrmation here.
 
|};
 
|};
 +
 +
<html>
 +
<p align="justify">In a cell, the total amount of ATP, ADP and  AMP molecules remains constant. Low glucose concentration results in high  activity of adenylate cyclase converting ATP into cAMP, who binds and converts  cAMP receptor protein (abbreviated as CRP) to DNA-binding configuration.  Conversely, when glucose concentration gets high, more ATP and less cAMP will  be produced, resulting in low DNA-binding  activity of CRP.</p>
 +
<p align="justify">We embed  gene cI of  lambda phage(BBa_P0451) downstream promoter PcstA (BBa_K118011) activated by the binding of CRP, and  genes of red fluorescence protein(RFP, BBa_I13507)  respectively downstream the promoter BBa_R0051  repressed by protein cI. In this way we construct an indirect regulation  pathway with sensus glucose, transcription activator CRP and transcription  repressor cI. If the device works as design, output of RFP will be increased following the elevation  of glucose concentration, and vice versa.</p>
 +
<p align="center"   <img src="https://static.igem.org/mediawiki/2012/3/38/Cellulose_1.png" width="500"
 +
height="421" hspace="2" vspace="1" align="middle" /></p>
 +
<p align="justify"><strong>Method</strong></p>
 +
<p align="justify"><strong>Construction of plasmid for indirect  regulation pathway</strong></p>
 +
<p align="justify">In this experiment, RFP reported the  function of the indirect regulation pathway.</p>
 +
<p align="justify">K861173: BBa_I13507, an mRFP generator with RBS and terminator, was  embedded after CRP activated promoter K118011.</p>
 +
<p align="justify">K861172: BBa_P0451, a cI generator with RBS and terminator,  was embedded after promoter BBa_K118011 activated by CRP.</p>
 +
<p align="justify">K861169:?  K861172 and I763007, a cI repressed RFP generator, were assembled .</p>
 +
<p align="justify">K861174: BBa_K137115, constitutively  expressing cI generator with promoter, RBS and terminator, was assembled  to  I763007.</p>
 +
<p align="justify">All new composite parts mentioned above  were transformed  to competent cells  of Escherichia coli str. DH5a. All positive clones are validated using PCR, restriction enzyme  digestion and DNA sequencing.</p>
 +
<p align="justify"><strong>Cell culture fluorescence measurement</strong></p>
 +
<p align="justify">Minimal medium with different concentration  of glucose(1mM, 4mM, 10 mM , 20 mM , 50 mM ,100 mM) were transferred into a  96-well plate, 200 μL for each well. Then each well was inoculated with 2 μL of  seed liquor which was activated overnight in M9 minimal medium with 50mM  glucose at 37℃. The wells  without inoculation were regarded as blank controls to revise the results.  Under  each condition, three parallel samples were setted. The plate was incubated  at  37℃, 150rpm. Cell  culture fluorescence was recorded on a SpectraMax M2 plate reader (Molecular  Devices). Excitation at 584 nm and emission at 607 nm were used. All  fluorescence was normalized with cell density by measuring the absorbance at 600  nm.</p>
 +
<p align="justify"><strong>Capturing  fluorescent  image </strong></p>
 +
<p align="justify">Cell morphology was observed  through fluorescence microscope, and the image of bacteria  with of each  glucose concentration were captured. To know more about these images, please  click on Here.</p>
 +
<p align="justify"><strong>Fluorescent analysis of cyto-imaging</strong></p>
 +
<p align="justify">A program named FANCY was ?designed to recognize single cell  and calculate the fluorescence strength  according to  the images.  For more  information, please click  Here.</p>
 +
<p align="justify"><strong>Results</strong></p>
 +
<p align="justify">Purified  plasmids  constructed before were digested  with XbaI and PstI for confirmation. The agarose gel electrophoresis showed  that the  lengths  were  correct. At last, the plasmids  were sent for sequencing. Results  showed  no mutation.</p>
 +
<p align="justify">In the cell culture fluorescence  measurement experiment, fluorescence of BBa_K861173 decreased  coordinating  with glucose  concentration, while BBa_K861169 was  reverse.The fluorescence of  BBa_K861174 was too low to record, so we do not  show it here. All of the results coincided with expected results indicating  that we have successfully constructed the promoter which was activated by high  concentration of glucose.</p>
 +
<p align="justify">Fluorescent  images indicated that all cells were growing normally, because the size and  morphology were both the same  as cells in LB  medium. The fluorescence of the cells in the images show the same discipline with  results from the fluorescence measurement experiments. <br>
 +
  The results of FANCY  are  showed as bellow, which conforms well  the results that showed above.</p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/9/9c/Indirect_Fig_1.png" width="506" height="409" alt=""></p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/d/dc/Indirect_Fig_2.png" width="500" height="220" alt=""></p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/2/21/Indirect_Fig_3.png" width="506" height="189" alt=""></p>
 +
<p align="justify"><img name="" src="https://static.igem.org/mediawiki/igem.org/c/c3/Indirect_Fig_4.png" width="500" height="226" alt=""></p>
 +
<p align="justify">&nbsp;</p>
 +
<p align="justify"><strong>Discussion </strong><br>
 +
</html)
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K861169 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K861169 EndReviews</partinfo>

Latest revision as of 03:35, 27 September 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_K861169

Device Design

In this design, we used promoter PcstA and an intermediate regulator cI, as PcstA is activated by CRP in low glucose concentration, whereas we need a device boosted by high concentration of glucose. As you can see, in this pathway, protein cI will be expressed when CRP is activated under low glucose concentration.The large amount of cI repressed the promoter R0051 heavily. On the contrary, when glucose concentration is high, the promoter is derepressed.

We made RFP as reporter and certify that the design works well via two different methods.

Fluorescence measurement

In the first method, Fluorescence intensity of cell culture was directly read from a plate reader. And all data is normalized by OD600.

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, indicating that the device works as expected.

The second method is practiced for single cells. The fluorescence pictures of different glucose concentration were captured by fluorescence microscope. Also, a program named FANCY was designed to recognize single cell and calculate the fluorescence strength in the images. As the result shows, it conformed well to that of the first method.

All these results indicated that the device we designed can meet the demand, promoters activated by glucose.

User Reviews

UNIQ9473f0cac5ebf760-partinfo-00000001-QINU UNIQ9473f0cac5ebf760-partinfo-00000002-QINU