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Team:Hannover/Background Plant Vector
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<h1>Background / Plant vector</h1> | <h1>Background / Plant vector</h1> | ||
| - | <p class="text">For our labwork we need a vector, which is proven to work while transferred into tobacco and Arabidopsis plants. Unfortunately iGEM does not provide a sequenced and saved one. Therefore we decided to establish such a vector in a sub-project.<br><br>We use the vector pORE_E3 <a href="http://www.ncbi.nlm.nih.gov/nuccore/AY562536">(AY562536.1)</a> and try to exchange the original pENTcup promoter into a 2x35s promoter because it’s better for our operations with tobacco and Arabidopsis plants. Additionally we want to change the origin MCS into an iGEM MCS – Berkeley <a href="http://parts.igem.org/Help:Standards/Assembly/RFC21">RFC[21]</a> assembly standard. RFC[21] allows cloning in frame as well as <a href="http://parts.igem.org/Help:Standards/Assembly/RFC12">RFC[12]</a>. RFC[12] has an decisive advantage over RCF[12]: the Prefix of RFC[21] consist of restriction sites without a GC-rich sequences of a NotI site. This is better | + | <p class="text">For our labwork we need a vector, which is proven to work while transferred into tobacco and Arabidopsis plants. Unfortunately iGEM does not provide a sequenced and saved one. Therefore we decided to establish such a vector in a sub-project.<br><br>We use the vector pORE_E3 <a href="http://www.ncbi.nlm.nih.gov/nuccore/AY562536">(AY562536.1)</a> and try to exchange the original pENTcup promoter into a 2x35s promoter because it’s better for our operations with tobacco and Arabidopsis plants. Additionally we want to change the origin MCS into an iGEM MCS – Berkeley <a href="http://parts.igem.org/Help:Standards/Assembly/RFC21">RFC[21]</a> assembly standard. RFC[21] allows cloning in frame as well as <a href="http://parts.igem.org/Help:Standards/Assembly/RFC12">RFC[12]</a>. RFC[12] has an decisive advantage <span id='a2'>over</span> RCF[12]: the Prefix of RFC[21] consist of restriction sites without a GC-rich sequences of a NotI site. This is better if you use a 5`UTR (often AT-rich) and increases GC-rich sequences are in general more deformable than AT-rich sequences. Our attempt is to use one pair of primers, which should hybridize in a water bath and thus build an insert, containing the MCS of RFC[21] and overhangs for cloning into pORE_E3.<br><br>Besides we want to remove the <span id='a1'>restriction</span> sites for XhoI and BglII from pORE_E3 because these shall not occur in iGEM’s vectors. With our modified pORE_E3_2x35s vector we want to provide iGEM a new standard for working with and in plants.</p> |
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Revision as of 17:02, 16 October 2014
Background / Plant vector
For our labwork we need a vector, which is proven to work while transferred into tobacco and Arabidopsis plants. Unfortunately iGEM does not provide a sequenced and saved one. Therefore we decided to establish such a vector in a sub-project.
We use the vector pORE_E3 (AY562536.1) and try to exchange the original pENTcup promoter into a 2x35s promoter because it’s better for our operations with tobacco and Arabidopsis plants. Additionally we want to change the origin MCS into an iGEM MCS – Berkeley RFC[21] assembly standard. RFC[21] allows cloning in frame as well as RFC[12]. RFC[12] has an decisive advantage over RCF[12]: the Prefix of RFC[21] consist of restriction sites without a GC-rich sequences of a NotI site. This is better if you use a 5`UTR (often AT-rich) and increases GC-rich sequences are in general more deformable than AT-rich sequences. Our attempt is to use one pair of primers, which should hybridize in a water bath and thus build an insert, containing the MCS of RFC[21] and overhangs for cloning into pORE_E3.
Besides we want to remove the restriction sites for XhoI and BglII from pORE_E3 because these shall not occur in iGEM’s vectors. With our modified pORE_E3_2x35s vector we want to provide iGEM a new standard for working with and in plants.
