BsubSecTagLP_5Bacillus subtilis Secretion Tag Library Plasmid 5 of 6
We design here the creation of Bacillus subtilis SecTag library plasmids (Bsub_SecTag_Lps). Each Bsub_SecTag_Lp has a pOpen-v3 backbone, and an insert consisting of a tandem array of many Bacillus subtilis secretion tag (SecTag) part variants, whose peptide sequences were compiled in Brockmeier et al. J. Mol Bio. 2006. 362(3), 392-402. Each SecTag is preceded by a custom RBS designed in RBScalc to maximize expression in Bacillus subtilis (assuming a RiboJ-type insulator upstream, which is not included on the library plasmid). Each RBS-SecTag pair is bounded by BsaI cut sites, with overhangs defining the tag as an ‘RBS/Loc’ part in the extended FreeGenes Protein Cloning (FG ProClo) assembly standard. Between each variant is 60bp of randomly generated DNA sequence to increase the sequence diversity and synthesizability of the library plasmid. And in the middle of each random DNA spacer is a PmeI blunt cut site, which can be cleaved during assembly reactions to reduce the chances of erroneous incorporation of two or more tandem RBS-SecTags into the finished plasmid. 148 RBS-SecTag variants are spread across 6 library plasmids.
37 C, shaking 300 rpm
Any Bacillus subtilis strain
BsaI required, PmeI optional
3' - TACT … CCAT - 5'