Team:Hong Kong HKU/cloning

Cloning

For pETES (Fig. 1), from upstream to downstream, notable features include (1) a T7 promoter, (2) a T7 RBS, (3) a His6 tag, (4) EutS in-frame with the His6 tag, (5) the remaining downstream components of BBa_K311004, where the Eut BMC structural components was derived from, (6) an additional RBS (sequence GAAGGAAG), (7) signal peptide for cargo localization, (8) a cargo fused with a signal peptide, or a multiple cloning site for cloning in any PCR products for BMC localization. For a detailed view of the constructed plasmids, readers can refer to the uploaded .dna files and view it via the online freeware SnapGene viewer.
Fig.1.

The construction region of the plasmids pETS, pETE, and pETES.
The signal peptide was made via a primer-dimer approach using long primers (~60bp). The EutSMNLK cluster was PCR-ed from the MiniPrep-ed product of BBa_K311004. For details in cloning methodology and experimental protocols, please refer to the Methods and Protocols page for reference.
The plasmids constructs were verified by Sanger sequencing, the data of which is available online as we have uploaded.