Team:Amsterdam/achievements/experimental

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As mentioned in our molecular design, the restriction enzyme is unable to cut methylated restriction sites. We can 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 memory module. Moreover, we expect to find either an off or intermediate state knowing that the LacH promoter driving the MTase has some basal activity.
As mentioned in our molecular design, the restriction enzyme is unable to cut methylated restriction sites. We can 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 memory module. Moreover, we expect to find either an off or intermediate state knowing that the LacH promoter driving the MTase has some basal activity.
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[[File:Amsterdam_experimental_results_1.jpg|700px]]
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[[File:Amsterdam_experimental_results_1.jpg|300px]]
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between an ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). On the other hand, incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Ultimately, a complete methylation of our memory module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between an ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). On the other hand, incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Ultimately, a complete methylation of our memory module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).

Revision as of 13:02, 26 September 2012