Team:Amsterdam/data/experimental

From 2012.igem.org

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<h4>Set up of the writer-reader module</h4>
<h4>Set up of the writer-reader module</h4>
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[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.
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<h4>Behavior of the writer-reader module under IPTG induction</h4>
<h4>Behavior of the writer-reader module under IPTG induction</h4>
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[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.
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Figure 2
Figure 2
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<h4>Behavior of the writer-reader module in varying growth conditions</h4>
<h4>Behavior of the writer-reader module in varying growth conditions</h4>
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[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 3]]
We aimed to characterize the activity of our writer during bacterial growth.  We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth.  
We aimed to characterize the activity of our writer during bacterial growth.  We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth.  
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[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 4]]
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Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader.   
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader.   
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The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer.  
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer.  
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[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 5]]
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Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results.  
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results.  
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<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4>
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4>
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[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]
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Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.

Revision as of 02:37, 27 September 2012