Difference between revisions of "Part:BBa J04430:Experience"

Revision as of 21:28, 16 October 2014

Applications of BBa_J04430

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.

Results

The fluorescence and OD of the cells was measured over 20 hours and at 4 different amounts of IPTG; 0μl, 4μl, 20μl, 100μl.

Graph showing fluorescence over time in in BL21 cells transformed with the IPTG inducible GFP plasmid and different amounts of IPTG including error bars in each case. This graph demonstrates that 4μl IPTG displays the greatest total fluorescence approximately 2.5 times greater than that with 0μl IPTG. All the cells had a greater fluorescence than that with 0μl indicating that it is essential for efficient GFP translation but at 20μl and 100μl total fluorescence is diminished.

This initially implies that the fluorescence is in fact reduced in the cells with more IPTG added, therefore we suspected there may be a toxic effect on the cells hence the OD was investigated.

Graph showing OD over fluorescence and demonstrates the suspected result; that IPTG reduced the growth of the BL21 cells during measurement over 4μl. At 20μl and 100μl the OD is reduced by approximately 50%, therefore must be taken into account when comparing efficacy of GFP expression.

This shows, most accurately, the comparison of the fluorescence of the BL21 cells with a spectrum of IPTG amounts added from 0μl to 100μl. This shows conclusively that with greater amounts of IPTG the fluorescence is increased. With 20μl and 100μl the fluorescence shows a steeper trajectory than at 4μl and with 0μl is significantly lowered. There is very little increase with 100μl from that seen with 100μl indicating that perhaps adding greater than 20μl is a waste of resources and has very little, or no, beneficial effect.

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>

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 <i>E. coli<i> 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.

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