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Team:Hannover/Background Plant Vector
From 2014.igem.org
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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< | + | <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='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> | ||
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Revision as of 14:22, 15 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[21]. 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.
