Team:Amsterdam/data/experimental

From 2012.igem.org

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[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]
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[[File:Amsterdam_exp_fig_14.png|300px|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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<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]]
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\>
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\>

Revision as of 03:32, 27 September 2012