B. subtilis Protein Secretion Toolkit

B. subtilis Protein Secretion Toolkit

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What is it

The efficient secretion of enzymes out of bacterial cells can greatly reduce the cost and complexity of enzyme production and purification. Bacillus subtilis is widely used in industrial protein manufacturing (e.g., enzymes) due to its Generally Regarded As Safe (GRAS) status and its ability to secrete enzymes tagged with the appropriate N-terminal peptide signal (SecTag).  We have designed a collection of 148 Bacillus subtilis SecTags that can be used to screen for efficient secretion of any given protein. 


Genetic Design and Construction

Each plasmid contains an array of many SecTag variants, each preceded by a B. subtilis ribosome binding site. RBS-SecTag pairs are bounded by BsaI restriction enzyme cut sites, and overhangs define each tag as an ‘RBS/Localization tag’ part in the extended Modular Cloning ‘MoClo’ assembly standard used for FreeGenes’s E. coli Protein Expression Toolkit. Parts have overhang syntax B and T1 (5'--TACT---part---CCAT--3') from the Protein Expression Toolkit. Between each variant are 60 randomly generated base pairs. In the middle of each random DNA spacer is a 8-base, blunt-cutting PmeI restriction site, which can be cleaved during assembly reactions to reduce the chance of erroneous incorporation of two or more tandem RBS-SecTags into any genetic construct built from these plasmids.


How to Use the Library

The B. subtilis SecTag library plasmids are designed for use in Golden Gate assembly reactions that attach both the RBS/SecTag pairs and a fluorescent protein (FP) tag to an protein of interest, assembling a library of FP-tagged enzymes with different SecTags in a single Golden Gate reaction. The assembled library can then be transformed into B. subtilis, and secretion can be screened for either by imaging a fluorescent ‘halo’ of secreted, diffusing protein around colonies on agar plates, or by high-throughput culturing of colonies in 96-deep-well plates, followed by centrifugation and measurement of secreted fluorescence via a plate reader.  The collection enables low-cost screening for efficient SecTag/enzyme pairs through a 1-pot Golden Gate reaction starting from only six or fewer SecTag variant plasmids. All plasmids are present on the plate in purified nucleic acid form. 

Instructions for Use:

Transform into E. coli Top10 cells or equivalent. Culture at 37C overnight, with shaking at around 300rpm. Check plate layout for selection marker. 

 

Where can I find more information

If you've already received the plate, check out the plate layout.

  1. This paper from Brockmeier et al. is where I got the amino acid sequences for the B. subtilis secretion tags.
  2. Salis lab’s RBS calculator is where the ribosome binding site for each secretion tag was designed.
  3. This collection is compatible with, and is sort of an extension of, the     FreeGenes E. coli Protein Expression Toolkit.

 

Teaser Image: All sub-images licensed by creative commons

Genes

Gene Name NCBI ID Freegenes ID
BsubSecTagLP_1 Bacillus subtilis Secretion Tag Library Plasmid 1 of 6 N/A BBF10K_000485
BsubSecTagLP_2 Bacillus subtilis Secretion Tag Library Plasmid 2 of 6 N/A BBF10K_000486
BsubSecTagLP_3 Bacillus subtilis Secretion Tag Library Plasmid 3 of 6 N/A BBF10K_000487
BsubSecTagLP_4 Bacillus subtilis Secretion Tag Library Plasmid 4 of 6 N/A BBF10K_000488
BsubSecTagLP_5 Bacillus subtilis Secretion Tag Library Plasmid 5 of 6 N/A BBF10K_000489
BsubSecTagLP_6 Bacillus subtilis Secretion Tag Library Plasmid 6 of 6 N/A BBF10K_000490

Download all of this information as a CSV from our GitHub.

Bionet

The bionet enables open peer-peer exchange of functional biomaterials and associated data. This product may also be available from bionet nodes that are more convenient to you. Here are other bionet nodes who may be willing to provide you this specific product.

Name Contact Country
Juan Sebastian Alvarez juan.alvarez1 {at} ucalgary {dot} ca Canada
Danielle Pedrolli danielle.pedrolli {at} unesp {dot} br Brazil

Download all of this information as a CSV from our GitHub.
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