http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&feed=atom&action=historyImperial/Infector Detector/Testing - Revision history2024-03-29T11:23:37ZRevision history for this page on the wikiMediaWiki 1.16.5http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=47045&oldid=prevMaira at 02:14, 27 October 20072007-10-27T02:14:50Z<p></p>
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</table>Mairahttp://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46566&oldid=prevJec105: /* Summary */2007-10-27T01:17:22Z<p><span class="autocomment">Summary</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key results of the testing were:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key results of the testing were:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*Several of the specifications have been met:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*Several of the specifications have been <ins class="diffchange diffchange-inline">tested and </ins>met:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**'''Input''':Sensitivity to 5-50nM AHL</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**'''Input''':Sensitivity to 5-50nM AHL</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**'''Operating Conditions''':25<sup>o</sup>C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**'''Operating Conditions''':25<sup>o</sup>C</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">**'''Response Time''':Response time to of visual threshold not determined, but peak fluorescence is reached at ~300 minutes.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Aims==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Aims==</div></td></tr>
</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46544&oldid=prevJec105: /* Summary */2007-10-27T01:15:11Z<p><span class="autocomment">Summary</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key results of the testing were:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key results of the testing were:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The optimum DNA concentration for [http://partsregistry.org/Part:BBa_T9002 '''pTet-LuxR-pLux-GFPmut3b'''] in our Commcercial S30 Cell extract is 4&micro;g.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Several of the specifications have been met:</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">**'''Input''':Sensitivity to 5-50nM AHL</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">**'''Operating Conditions''':25<sup>o</sup>C</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Aims==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>== Aims==</div></td></tr>
</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46519&oldid=prevJec105: /* AHL Testing */2007-10-27T01:12:59Z<p><span class="autocomment">AHL Testing</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|style="text-align: left;" |<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|style="text-align: left;" |<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1.3 shows us the following:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1.3 shows us the following:</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Functional at 25<sup>o</sup>C</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The output of '''GFPmut3b increases with input of AHL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The output of '''GFPmut3b increases with input of AHL'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The system is sensitive to a range of '''5-1000nM AHL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The system is sensitive to a range of '''5-1000nM AHL'''</div></td></tr>
</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46485&oldid=prevJec105: /* DNA Concentrations */2007-10-27T01:09:34Z<p><span class="autocomment">DNA Concentrations</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{|align="center" style="text-align: center; border-top:1px solid #000077; border-right:1px solid #000077; border-bottom:1px solid #000077; border-left:1px solid #000077;" </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{|align="center" style="text-align: center; border-top:1px solid #000077; border-right:1px solid #000077; border-bottom:1px solid #000077; border-left:1px solid #000077;" </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' - The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|<br><center>[[Image:IC 2007 DNA Concentration.PNG|thumb|420px|left|Fig.1.1:Molecules of GFPmut3b synthesised over time, for each DNA Concentration ''in vitro'' <ins class="diffchange diffchange-inline">at 25<sup>o</sup>C </ins>- The fluorescence was measured over time for each experiment and converted into molecules of GFPmut3b ''in vitro'' </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Imperial/Wet Lab/Results/Res1.3/Converting_Units| using our calibration curve]] Click here for [[Imperial/Wet_Lab/Results/ID2.1| results]] and [[Imperial/Wet_Lab/Protocols/ID2.1| protocol]].]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes.<br><br><br>]] </center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[image:IC 2007 DNA Concentration 360mins.PNG|thumb|420px|right|Fig.1.2:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes <ins class="diffchange diffchange-inline">at 25<sup>o</sup>C</ins>.<br><br><br>]] </center></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|-</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|style="text-align: left;" |<br>The Results above show that the optimum DNA concentration for ''in vitro'' is 4&micro;g for 50nM AHL. From figure 1.1. and 1.2 it can be seen that as DNA concentration increases above 4&micro;g the GFPmut3b molecules synthesised decrease. Interestingly for figure 1.2 the graph can be split into several regions of how the DNA concentration changes the output of GFPmut3b synthesis:</div></td></tr>
</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46482&oldid=prevJec105: /* AHL Testing */2007-10-27T01:08:47Z<p><span class="autocomment">AHL Testing</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|420px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>|<br><center>[[Image:GFPMolecule syn ID2 Final.PNG|thumb|center|420px|left|Fig.1.3: Molcules of GFPmut3b synthesised vs AHL concentrations <ins class="diffchange diffchange-inline">at 25<sup>o</sup>C</ins>. The Testing was carried out for the 4&micro;g of DNA. Again the fluorescence reading was converted into GFPmut3b molecules via the calibration curve. Click for full results and protocols can be found on the links [[Imperial/Wet Lab/Results/ID3.1| results]] and [[Imperial/Wet_Lab/Protocols/ID3.1|protocol]] pages.]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[[Image:Titrations curve - molecules.PNG|thumb|440px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[[Image:Titrations curve - molecules.PNG|thumb|440px|right|Fig.1.4:Molecules of GFPmut3b synthesised for each DNA Concentration ''in vitro'', after 360 minutes <ins class="diffchange diffchange-inline">at 25<sup>o</sup>C</ins>. The data is from the fluorescence measurement at 360minutes which was converted to GFPmut3b molecules and plotted against [AHL]. The scale on the X axis is a logarithmic scale.]]</center></div></td></tr>
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</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46434&oldid=prevJec105: /* AHL Testing */2007-10-27T01:00:15Z<p><span class="autocomment">AHL Testing</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The '''lower threshold of response''' is the AHL concentration that the construct will respond</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The '''lower threshold of response''' is the AHL concentration that the construct will respond</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The '''upper threshold of response''' is the value of AHL the system is saturated and increasing AHL will not increase the rate of GFP synthesis.<br><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">For more detailed analysis please see the [[Imperial/Wet_Lab/Results/ID3.1| Results page]]</del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
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</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46426&oldid=prevJec105: /* AHL Testing */2007-10-27T00:59:05Z<p><span class="autocomment">AHL Testing</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The output of '''GFPmut3b increases with input of AHL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The output of '''GFPmut3b increases with input of AHL'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The system is sensitive to a range of '''5-1000nM AHL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The system is sensitive to a range of '''5-1000nM AHL'''</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced<del class="diffchange diffchange-inline">, showing </del>that the LuxR under the control of pTet is the major source of energy consumption. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR. <br><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*The GFPmut3b molecules synthesis stops at '''~300minutes'''. This could be due to steady state or due to no synthesis of GFPmut3b. It is known not to be steady state because the [[Imperial/Wet_Lab/Results/Res1.4| degradation experiment]] proved degradation is negligible. Interestingly this time at which synthesis stops is independent of the GFPmut3b molecules produced <ins class="diffchange diffchange-inline">as all the [AHL] level off at the same time. This shows </ins>that the LuxR under the control of pTet is the major source of energy consumption<ins class="diffchange diffchange-inline">. The pTet is a very strong promoter and is a big consumer of the energy of the chassis</ins>. This highlights the advantages of using the construct 2 [http://partsregistry.org/Part:BBa_J37032 pLux-GFPmut3b] that does not have the energetic burden of producing LuxR <ins class="diffchange diffchange-inline">under pTet</ins>. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1.4 shows us the following:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Figure 1.4 shows us the following:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*The shape of the '''Transfer function''' shows a linear range of response between 5nM and 100nM AHL. This defines the thresholds of response.</div></td></tr>
</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46405&oldid=prevJec105: /* DNA Concentrations */2007-10-27T00:55:38Z<p><span class="autocomment">DNA Concentrations</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*'''Inhibition phase'''- Increasing the DNA concentration actually inhibits the rate of protein synthesis.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]</del><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Although there is only a limited number of data points and there is a lot of error we feel that the results do show that 4&micro;g was taken as the maximum and used for the rest of the testing. This agrees with the instructions provided by Promega which state that above 4&micro;g the rate of protein synthesis is inhibited.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Although there is only a limited number of data points and there is a lot of error we feel that the results do show that 4&micro;g was taken as the maximum and used for the rest of the testing. This agrees with the instructions provided by Promega which state that above 4&micro;g the rate of protein synthesis is inhibited.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
</table>Jec105http://2007.igem.org/wiki/index.php?title=Imperial/Infector_Detector/Testing&diff=46395&oldid=prevJec105: /* DNA Concentrations */2007-10-27T00:53:49Z<p><span class="autocomment">DNA Concentrations</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The fact that increasing DNA concentration above 4&micro;g causes a decrease in rate of protein synthesis is very interesting. The reason for this is thought to be that increasing DNA concentration causes problems with premature translational termination. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For more information on the results please go the the [[Imperial/Wet_Lab/Results/ID2.1| results page]]<br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">The </del>4&micro;g was taken as the maximum and used for the rest of the testing.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Although there is only a limited number of data points and there is a lot of error we feel that the results do show that </ins>4&micro;g was taken as the maximum and used for the rest of the testing<ins class="diffchange diffchange-inline">. This agrees with the instructions provided by Promega which state that above 4&micro;g the rate of protein synthesis is inhibited</ins>.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>|}</div></td></tr>
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</table>Jec105