Team:Amsterdam/data/experimental

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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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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 the ‘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). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).
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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 the ‘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). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).
<h4>Behavior of the writer-reader module under IPTG induction</h4>
<h4>Behavior of the writer-reader module under IPTG induction</h4>

Revision as of 03:37, 27 September 2012