Team:Amsterdam/data/experimental

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<h1>Experimental results</h1>
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<h1>Experimental Results</h1>
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<h2>Functionality of the writer-reader module under different sensor modules</h2>
<h2>Functionality of the writer-reader module under different sensor modules</h2>
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We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in in Library Efficient® DH5α™ competent cells (Invitrogen).
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We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).
<h4>Set up of the writer-reader module</h4>
<h4>Set up of the writer-reader module</h4>
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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>
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The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.
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[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 10]]
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[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]
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[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.
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Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.
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Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase (figure 10).
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Over the course of time the methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\>
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Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose (figure 11). This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\>
<h2>Towards the Cellular Logbook</h2>
<h2>Towards the Cellular Logbook</h2>
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[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 11]]
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[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]
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[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 12]]
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[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]
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After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 11 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 12). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\>
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After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\>
<h1>Reference List</h1>
<h1>Reference List</h1>

Latest revision as of 03:52, 27 September 2012