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Team:Warwick/Parts/bb
From 2014.igem.org
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<h5> Experimental Design</h5> | <h5> Experimental Design</h5> | ||
| - | <p><b>Test:</b> GFP expression rate in cells co-transformed with plasmid that contains the promoter and a reversed GFP sequence and a plasmid containing the RdRP (Part:BBa_K1442100).</p> | + | <!--<p><b>Test:</b> GFP expression rate in cells co-transformed with plasmid that contains the promoter and a reversed GFP sequence and a plasmid containing the RdRP (Part:BBa_K1442100).</p> |
<p><b>Positive Control:</b> GFP expression rate in cells transformed with Biobrick plasmid that contains a gene for GFP with an inducible T7 promoter (Part:BBa_J04430). Promoter induction strength in response to different concentrations of added inducer (IPTG) was characterised.</p> | <p><b>Positive Control:</b> GFP expression rate in cells transformed with Biobrick plasmid that contains a gene for GFP with an inducible T7 promoter (Part:BBa_J04430). Promoter induction strength in response to different concentrations of added inducer (IPTG) was characterised.</p> | ||
<p><b>Negative Control:</b> GFP expression rate in cells co-transformed with the Promoter Testing Module and a plasmid containing a mutated version of the RdRP (Part:BBa_K1442101) that has been proven to be unable to direct successful replication of RNA.</p> | <p><b>Negative Control:</b> GFP expression rate in cells co-transformed with the Promoter Testing Module and a plasmid containing a mutated version of the RdRP (Part:BBa_K1442101) that has been proven to be unable to direct successful replication of RNA.</p> | ||
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<p>As the plotted graphs for each IPTG concentration do not show a plateau at any of the time points investigated we compared the gradient of the graphs to indicate the relative potential for fluorescence under each condition. <p> | <p>As the plotted graphs for each IPTG concentration do not show a plateau at any of the time points investigated we compared the gradient of the graphs to indicate the relative potential for fluorescence under each condition. <p> | ||
<p><img src="https://static.igem.org/mediawiki/parts/5/52/Bar_chart_FoverOD.PNG"/></p> | <p><img src="https://static.igem.org/mediawiki/parts/5/52/Bar_chart_FoverOD.PNG"/></p> | ||
| - | <p><i>This bar chart compares the gradient of the lines plotted in the graph above in order to examine the relative potential of the transformed cells under the varied conditions. Only values obtained between 0.3 and 0.5 OD units were included in the creation of this chart as this is the OD between which ''E. coli'' demonstrates its exponential growth phase and would be expressing the transformed plasmid. This shows that the fluorescence increases exponentially from just over 1000 arbitrary units in cells with 0μl IPTG added to over 6 times as much relative fluorescence in those cells with 20μ added. However, this bar chart also demonstrates the same plateau effect, as seen in the line graph above, and supports the hypothesis that above 20μl more IPTG has no beneficial effect and is a waste of resources.</i></p> | + | <p><i>This bar chart compares the gradient of the lines plotted in the graph above in order to examine the relative potential of the transformed cells under the varied conditions. Only values obtained between 0.3 and 0.5 OD units were included in the creation of this chart as this is the OD between which ''E. coli'' demonstrates its exponential growth phase and would be expressing the transformed plasmid. This shows that the fluorescence increases exponentially from just over 1000 arbitrary units in cells with 0μl IPTG added to over 6 times as much relative fluorescence in those cells with 20μ added. However, this bar chart also demonstrates the same plateau effect, as seen in the line graph above, and supports the hypothesis that above 20μl more IPTG has no beneficial effect and is a waste of resources.</i></p>--> |
Revision as of 06:26, 17 October 2014
EXISTING BIOBRICK CHARACTERISATION
As part of the criterion for the Gold medal, we set about trying to improve the characterisation of an existing part on the registy. In line with the rest of our project, we felt it pertinent that we characterise something relevant and as such we chose this part. The information below is taken from the registry page:
Introduction
Warwick iGEM 2014 used this part as a positive control for our experiments investigating promoter strengths for a self replicating RNA strand, using the Hepatitis C Virus NS5B gene encoding RNA dependent RNA polymerase, as the replicating enzyme.
This was measured using a tecan Magellan plate reader in which fluorescence in the GFP range was measured over a period of 24 hours with both biological and technical replicates for each transformed cell type.
