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From 2014.igem.org

Results / Plant vector

We want to provide iGEM a new Plant vector with a BioBrick RFC[21]MCS and 2x35S promotor.

• exchange the original promoter enTCUP2 of the binary vector pORE_E3 (AY562536.1) into a 2x35s promoter via restriction site based cloning using these protocols
• removal of restriction sites in our pORE_E3_2x35S
• cloning the RFC[21]MCS into this vector

Results

The exchange of the original promoter of the pORE_E3 vector was successful. We gained a “new” vector with a 2x35s promoter (pORE_E3_2x35s). Furthermore we were able to remove the restriction sites XhoI and BglII from this vector. This was checked by a double digest and sequencing. Unfortunately our last attempt, changing the MCS to the MCS of RFC[21] via the insertion of two hybridized primers with overhangs, failed several times. Even a modified protocol couldn’t bring us the expected success. At the end, we ran out of time.

Of cause, we could have synthesized the insert, cut it with restriction enzymes and then clone it into the vector, but that’s what everyone could have done…

Fig. 1: Pictures showing the vector pORE_E3 (left) with the origin sequence and (right) our designed PORE_2x35S_RCF[21] with a 2x35s promoter and the MCS of RFC[21] (plasmid maps originally taken from www.snapgene.com/resources).