Team:Manchester-Graz/Project/Experiments
Experiments
Vector assembly:
Fig.1 Vector assembly strategy. [A] all gBlocks are cloned in pJet. [B]
After sequence verifying the gBlocks are cut out of pJet using restriction sites
in the flanking region of each gBlock, [C] With overlap extention PCR two or
three gBlocks are assembled to a bigger fragment. [D] The resulting product is
cloned in pJet, secquence verified and cut out again using restriction sites.
[E] Using Gibson assembly the fragments are assembled to a full vector [F].
Our vector pCERI is assembled from scratch, by ordering synthesized genes on gBlocks that are needed for its functionality. All the gBlocks we ordered from IDT already contain overlapping sequences to the following gBlock. Before we actually want to assemble our vector however, we want to make sure the used gBlocks have the right sequence. Thus all gene fragments are blunt-end cloned into the commercial vector pJET 1.2 for sequencing purpose (Fig.1 A). Sequence verified gBlocks are afterwards cut out with flanking restriction sites on each gBlock, so no additional bases from pJET 1.2 that would disturb the following overlap-extension PCR (OE-PCR) remain (Fig.1 B).
After sequence verification two to three gBlocks are fused to larger DNA fragments by OE-PCR (Fig.1 C). These fragments are also cloned into pJET 1.2 and cut out again after sequence verification (Fig.1 D). The fragment bla_p15A, containing the p15A origin of replication is cloned into another commercial vector pPIC9 via two flanking BglII restriction sites to circumvent problems with two bacterial origins of replications on one plasmid (not shown). The four resulting fragments, PaidA_mRFP, PesaRC_CFP_CepI, EsaI_CepR_EsaR_PesaS and p15A_bla are fused to a functional circular vector by Gibson assembly (Fig.1 E).
