BsubSecTagLP_2

Bacillus subtilis Secretion Tag Library Plasmid 2 of 6
BBF10K_000486
source
Bacillus subtilis

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.

attr.
Isaac Larkin

Usage

growth

shipping strain
Escherichia coli Top10
growth conditions
37 C, shaking 300 rpm
antibiotic
ampicillin

expression

strain
Any Bacillus subtilis strain
promoter
N/A
inducer
N/A

cloning

method
Golden Gate
enzyme
BsaI required, PmeI optional
overhangs
3' - TACT … CCAT - 5'

sequencing

forward primer
M13 forward
reverse primer
M13 reverse

Construct

plasmid name
No Value
plasmid size
No Value
insert size
4962
origin
ColE1
copy number
500-700

Safety

BSL
BSL1

other information

No Value

References

primary references

NCBI
UniProt
Available Elsewhere
No Value

protocols and instructions

No Value

citations

No Value

intellectual property

We are unaware of third-party property rights claims on uses of this item as of we.