Bee Microbiome Toolkit

The Bee Microbiome Toolkit (BTK) is a Golden-Gate compatible toolkit of genetic parts designed for working with newly isolated, non-model bacteria. Like in the yeast toolkit, type IIS enzymatic assembly of parts with defined overhangs create complete vectors. Plasmid elements— promoters, coding sequences, antibiotic resistance genes— can be independently replaced. The toolkit includes broad-host-range promoters and a broad-host-range plasmid backbone that replicates 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 soon be available from Addgene. If you use the BTK parts or these protocols in your work, please cite: 1. Leonard, S. P. et al. Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids. ACS Synth. Biol. acssynbio.7b00399 (2018). doi:10.1021/acssynbio.7b00399.

BTK Protocols

Design a new part Design overhangs for the amplification of a new custom part to be assembled into a part plasmid.

Assemble a new part plasmid Assemble a new custom part plasmid following amplification with part specific overhangs.

Build a first-stage 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.

Build a second-stage plasmid Assemble multiple first-stage transcriptional unit plasmids.

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.