Broad-Host-Range Toolkit

The Broad-host-range toolkit (BTK) is a Golden-Gate compatible toolkit of genetic parts designed for working with newly isolated, non-model bacteria. Like the yeast toolkit, it relies on type IIS enzymatic assembly of Type 1-8 parts with defined nucleotide overhangs into complete vectors. The toolkit includes broad-host-range (BHR) promoters and a BHR plasmid backbone that is replicated in diverse Gram-negative and Gram-positive hosts. BTK plasmids possess an origin of transfer (oriT), and can be delivered by conjugation or electroporation. Here we describe example protocols and use cases of the BTK. BTK parts will be deposited and available from Addgene. If you use the BTK parts or these protocols in your work, please cite: .

Detailed description of BTK components and Parts List

BTK Protocols

Build a plasmid from existing BTK parts If the BTK contains all the parts you need, you can build a complete plasmid with a single BsaI reaction.

Make a new part If you want to include a new coding sequence or promoter or some other custom part, follow these instructions to assemble a new part plasmid.

Build a second-stage plasmid

Build a plasmid for CRISPRi gene suppression

Build a plasmid for disruption or knockout of bacterial genes

Deliver plasmids via conjugation into new hosts

(old old old)

Making a new Golden Gate Part Vector
(original, detailed version) Cloning into the entry vector.

Assembly of Cassette Plasmids

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Topic revision: r2 - 14 Sep 2017 - 14:47:49 - Main.SeanLeonard
 
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