Difference between revisions of "Part:BBa K1442024:Experience"
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<p><html><body><img src="https://static.igem.org/mediawiki/parts/4/49/Table_IRES.PNG"/></body></html></p>
 
<p><html><body><img src="https://static.igem.org/mediawiki/parts/4/49/Table_IRES.PNG"/></body></html></p>
 
<p><i>We measured fluorescence using a tecan Magellan plate reader at wavelength 465nm - 530nm (that corresponding to GFP). Each IRES had ten replicates in each cell and hence error bars had an accuracy of 0.1. The fluorescence of media and cells themselves were accounted for in each case and the negative control used was the human optimised forwards GFP <html><body><a href="https://parts.igem.org/Part:BBa_K1442106">part:BBa_K1442106</a></body></html> without IRES included.</i></p>
 
<p><i>We measured fluorescence using a tecan Magellan plate reader at wavelength 465nm - 530nm (that corresponding to GFP). Each IRES had ten replicates in each cell and hence error bars had an accuracy of 0.1. The fluorescence of media and cells themselves were accounted for in each case and the negative control used was the human optimised forwards GFP <html><body><a href="https://parts.igem.org/Part:BBa_K1442106">part:BBa_K1442106</a></body></html> without IRES included.</i></p>
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<p><html><body><img src="https://static.igem.org/mediawiki/parts/0/05/Bar_chart_all_IRES_with_error_bars.PNG"/></body></html>
 
<p></p>
 
<p></p>
 
<p><b>HeLa cell experimentation</b></p>
 
<p><b>HeLa cell experimentation</b></p>
 
<p>In HeLa cells the NKRF IRES have previously been directly compared with firefly luciferase association in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#__ffn_sectitle Oumard et al 2000]</p>
 
<p>In HeLa cells the NKRF IRES have previously been directly compared with firefly luciferase association in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#__ffn_sectitle Oumard et al 2000]</p>
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<p>The
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<p>[[File:https://static.igem.org/mediawiki/parts/f/fd/IRES_in_Hela.PNG]]</p>
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<p>This clearly indicates that the NKRF IRES is significantly more efficient at initiating translation of GFP following this IRES conferring 2.4 times greater intensity than EMCV. Strangely the negative control showed higher fluorescence than the EMCV IRES also. This may have been due to increased growth rate of the cells or slower depletion of the growth media hence increasing overall fluorescence. Due to time restraints and limited number of wells in the plate we did not have as many replicates with this control.</p>
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<p><b>Huh 7.5 experimentation</b></p>
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<p>This is, to our knowledge, the first
  
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<p><html><body><img src="https://static.igem.org/mediawiki/parts/0/05/Bar_chart_all_IRES_with_error_bars.PNG"/></body></html>
 
  
 
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Revision as of 16:13, 11 October 2014

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.

Experimentation

We compared efficiency of EMCV and NKRF in both HeLa and Huh 7.5 cells. EMCV and HeLa have been previously investigated in HeLa cells however have not been directly compared in Huh 7.5 cells. We compared each of these at the peak of fluorescence for most of the cells, 1800 seconds after loading. Due to the nature of the measurement equipment we were unable to supply CO2 to the cells during measurement and the cells gradually died during the 25 hour measurement period explaining the exponential decline in fluorescence.

This shows an exponential decline as the cells die due to lack of CO2 supply and shows the same overall trend in all the cells with maximal fluorescence occurring at approximately 1800 seconds

The fluorescence of each type of transfected cell was taken at 1800 seconds and averaged across the groups while fluorescence was adjusted for media and cell alone fluorescence and consolidated in this table.

We measured fluorescence using a tecan Magellan plate reader at wavelength 465nm - 530nm (that corresponding to GFP). Each IRES had ten replicates in each cell and hence error bars had an accuracy of 0.1. The fluorescence of media and cells themselves were accounted for in each case and the negative control used was the human optimised forwards GFP part:BBa_K1442106 without IRES included.

<p>

HeLa cell experimentation

In HeLa cells the NKRF IRES have previously been directly compared with firefly luciferase association in [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC85491/#__ffn_sectitle Oumard et al 2000]

File:Https://static.igem.org/mediawiki/parts/f/fd/IRES in Hela.PNG

This clearly indicates that the NKRF IRES is significantly more efficient at initiating translation of GFP following this IRES conferring 2.4 times greater intensity than EMCV. Strangely the negative control showed higher fluorescence than the EMCV IRES also. This may have been due to increased growth rate of the cells or slower depletion of the growth media hence increasing overall fluorescence. Due to time restraints and limited number of wells in the plate we did not have as many replicates with this control.

Huh 7.5 experimentation

This is, to our knowledge, the first UNIQ364a5da57adc7ca4-partinfo-00000004-QINU UNIQ364a5da57adc7ca4-partinfo-00000005-QINU