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Team:Hannover/Background Plant Vector
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| - | <h1>Background / Plant vector</h1> | + | <h1><a href="https://2014.igem.org/Team:Hannover/Background_Project">Background</a> / 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 | + | <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 used the vector pORE_E3 <a href="http://www.ncbi.nlm.nih.gov/nuccore/AY562536">(AY562536.1)</a> and tried to exchange the original enTCUP2 promoter into a 2x35s promoter because it was better for our operations with tobacco and Arabidopsis plants. Additionally we wanted 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[21] has an decisive advantage <span id='a2'>over</span> RCF[12]: the prefix of RFC[21] consists of only two restriction sites and has no GC-rich sequence of the NotI-restriction-site (RFC[12] has a NotI-site). A missing NotI-site is better if you use a 5`UTR and in general, GC-rich sequences are more easily deformable than AT-rich sequences. Our attempt was 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 wanted 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 wanted to provide iGEM a new standard for working with and in plants.</p> |
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| - | <div class='annotation' ref='a1'>You find all forbidden restriction sites <a href="http://parts.igem.org/Help:Protocol/DNA_Synthesis">here</a></div> | + | <div class='annotation' ref='a1'>You can find all forbidden restriction sites <a href="http://parts.igem.org/Help:Protocol/DNA_Synthesis">here</a></div> |
<div class='annotation' ref='a2'>An alternative method is to synthesize the insert, cut it with restriction enzymes and then clone it into the vector.</div> | <div class='annotation' ref='a2'>An alternative method is to synthesize the insert, cut it with restriction enzymes and then clone it into the vector.</div> | ||
</div> | </div> | ||
Latest revision as of 21:44, 17 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 used the vector pORE_E3 (AY562536.1) and tried to exchange the original enTCUP2 promoter into a 2x35s promoter because it was better for our operations with tobacco and Arabidopsis plants. Additionally we wanted 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[21] has an decisive advantage over RCF[12]: the prefix of RFC[21] consists of only two restriction sites and has no GC-rich sequence of the NotI-restriction-site (RFC[12] has a NotI-site). A missing NotI-site is better if you use a 5`UTR and in general, GC-rich sequences are more easily deformable than AT-rich sequences. Our attempt was 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 wanted 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 wanted to provide iGEM a new standard for working with and in plants.
