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.