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Team:Hannover/Background pASK
From 2014.igem.org
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| - | <h1>Background / Heterologoues expression in <i>Escherichia coli</i></h1> | + | <h1><a href="https://2014.igem.org/Team:Hannover/Background_Project">Background</a> / Heterologoues expression in <i>Escherichia coli</i></h1> |
| - | <p class="text"> | + | <p class="text">To characterize 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 | + | 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 the Origami 2 strain, which is optimized to form 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 |
| - | <p class="text">We decided to use Tags | + | " 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 to easily purify our protein. Therefore we used the pASK vector, because it delivers a His- and a Strep-Tag sequence. Moreover we modified the pASK plasmid with enterokinase sites (N- and C-terminus). Via enzymatically digest we are able to to get rid of the disturbing Tag regions.<br><br>Beside the His- and <span id='a2'>Strep-Tag</span>, pASK contains an anhydrotetracyclin-promotor (tet-promotor) for the induction of the protein expression. In contrast to the lac-promotor (inducible via e.g. lactose) the tet-promotor does not need a high concentration of anhydrotetracyclin to be induced. Furthermore it is highly regulated and shows only a low background activity.</p> | ||
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| - | <div class='annotation' ref='a1'>An alternative method is the transformation of tobacco | + | <div class='annotation' ref='a1'>An alternative method is the transformation of tobacco plants (<i>Nicotiana tabacum</i>). Protein production in an eucaroytic organism enhances the formation of disulfide bonds. Greetings from us... the plant scientists!</div> |
| - | <div class='annotation' ref='a2'>Remember | + | <div class='annotation' ref='a2'>Remember that protein purification via chromatography is very complicated! Use Tags because it’s easier and faster.</div> |
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Latest revision as of 13:27, 17 October 2014
Background / Heterologoues expression in Escherichia coli
To characterize 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 the Origami 2 strain, which is optimized to form disulfide bonds.
The Use of the pASK-Vector
We decided to use Tags to easily purify our protein. Therefore we used the pASK vector, because it delivers a His- and a Strep-Tag sequence. Moreover we modified the pASK plasmid with enterokinase sites (N- and C-terminus). Via enzymatically digest we are able to to get rid of the disturbing Tag regions.
Beside the His- and Strep-Tag, pASK contains an anhydrotetracyclin-promotor (tet-promotor) for the induction of the protein expression. In contrast to the lac-promotor (inducible via e.g. lactose) the tet-promotor does not need a high concentration of anhydrotetracyclin to be induced. Furthermore it is highly regulated and shows only a low background activity.
An alternative method is the transformation of tobacco plants (Nicotiana tabacum). Protein production in an eucaroytic organism enhances the formation of disulfide bonds. Greetings from us... the plant scientists!
Remember that protein purification via chromatography is very complicated! Use Tags because it’s easier and faster.
