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Team:Hannover/Background pASK
From 2014.igem.org
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<p class="text">For the characterization of our metalbinding protein (T4MBP), it was necessary to produce big quantities of it. | <p class="text">For the characterization of our metalbinding protein (T4MBP), it was necessary to produce big quantities of it. | ||
For us the method of choice was to use the bacteria <i>Escherichia coli</i> (<i>E. coli</i>). Poorly <i>E. coli</i> is bad in forming disulfide bonds. Because or T4MBP consists of several <span id='a1'>disulfide bonds</span> we decided to use Origami2 strain, which is optimized for forming disulfide bonds.</p> | For us the method of choice was to use the bacteria <i>Escherichia coli</i> (<i>E. coli</i>). Poorly <i>E. coli</i> is bad in forming disulfide bonds. Because or T4MBP consists of several <span id='a1'>disulfide bonds</span> we decided to use Origami2 strain, which is optimized for forming disulfide bonds.</p> | ||
| - | <h2>The Use of the pASK-Vector</h2> | + | <h2>The Use of the pASK-Vector</h2><a href="https://static.igem.org/mediawiki/2014/b/bc/HAnnover_20141015_Skizze16.jpg |
| + | " data-lightbox="galery" data-title="Bacteria"><img src="https://static.igem.org/mediawiki/2014/b/bc/HAnnover_20141015_Skizze16.jpg | ||
| + | " width="150px" style="float:left"></a> | ||
<p class="text">We decided to use Tags for easier protein purification. Therefor we used the pASK vector, because he delivers a His- and a Strep-Tag sequence. Moreover we modified the pASK plasmid with enterokinase sites (N- and C-terminus). Via enzymatically digest it would be possible to get rid of disturbing Tag regions.<br><br>Beside the His- and <span id='a2'>Strep-Tag</span>, pASK contains an anhydrotetracyclin-promotor for the induction of the protein expression. In contrast of the lac-Promotor the Tet-Promotor does not need a high concentration of the inducer. Furthermore the Tet-promotor is highly regulated and shows only low background activity.</p> | <p class="text">We decided to use Tags for easier protein purification. Therefor we used the pASK vector, because he delivers a His- and a Strep-Tag sequence. Moreover we modified the pASK plasmid with enterokinase sites (N- and C-terminus). Via enzymatically digest it would be possible to get rid of disturbing Tag regions.<br><br>Beside the His- and <span id='a2'>Strep-Tag</span>, pASK contains an anhydrotetracyclin-promotor for the induction of the protein expression. In contrast of the lac-Promotor the Tet-Promotor does not need a high concentration of the inducer. Furthermore the Tet-promotor is highly regulated and shows only low background activity.</p> | ||
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<div id='side_content'> | <div id='side_content'> | ||
<div class='annotation' ref='a1'>An alternative method is the transformation of tobacco plant (Nicotiana tabacum). Protein production in an eucaroytic organism enhances the formation of disulfide bonds. Greetings from us... the plant scientists</div> | <div class='annotation' ref='a1'>An alternative method is the transformation of tobacco plant (Nicotiana tabacum). Protein production in an eucaroytic organism enhances the formation of disulfide bonds. Greetings from us... the plant scientists</div> | ||
Revision as of 20:46, 15 October 2014
Background / Heterologoues expression in Escherichia coli
For the characterization of our metalbinding protein (T4MBP), it was necessary to produce big quantities of it. For us the method of choice was to use the bacteria Escherichia coli (E. coli). Poorly E. coli is bad in forming disulfide bonds. Because or T4MBP consists of several disulfide bonds we decided to use Origami2 strain, which is optimized for forming disulfide bonds.
The Use of the pASK-Vector
We decided to use Tags for easier protein purification. Therefor we used the pASK vector, because he delivers a His- and a Strep-Tag sequence. Moreover we modified the pASK plasmid with enterokinase sites (N- and C-terminus). Via enzymatically digest it would be possible to get rid of disturbing Tag regions.
Beside the His- and Strep-Tag, pASK contains an anhydrotetracyclin-promotor for the induction of the protein expression. In contrast of the lac-Promotor the Tet-Promotor does not need a high concentration of the inducer. Furthermore the Tet-promotor is highly regulated and shows only low background activity.
