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Lab Protocols

Plasmids

Below are the current set of Coleman lab plasmids. Click the image to view in Addgene.

pUS252

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This has the ‘original’ free use GFP (fuGFP) gene cloned into the pK18 vector (KmR, high copy, E.coli only), where it is constitutively expressed. This GFP absorbs primarily long-wave UV, and emits green.

pMycoFos

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This is a shuttle/expression fosmid, that replicates in E.coli and Mycobacterium, and gives KmR in both genera. It can be used to clone large DNA inserts (up to ~40 kb) at the multiple cloning site (EcoRI, PacI, NheI, SwaI, BamHI), and the resultant large plasmids can be packaged in phage lambda heads for transfection of E.coli. It has an acetamide-inducible promoter for cloned genes that functions in Mycobacterium but not E.coli. The plasmid can be induced from low copy to high copy number in E.coli using arabinose. It is low-copy number in Mycobacterium.

pUS116

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This is a shuttle/expression plasmid, that replicates in E.coli and Mycobacterium, and gives KmR in both genera. Genes cloned at the multiple cloning site (NdeI, BamHI, EcoRI, NheI) are expressed from the acetamide-inducible promoter in Mycobacterium. The plasmid is high copy number in E.coli and low copy number in Mycobacterium.

pUS232

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This is a broad host range expression plasmid that will replicate in many Gram negative genera. It is based on the low-copy-number pBBR replicon, and gives KmR. The plasmid is designed to allow cloning of genes of interest using one restriction site in the left MCS and one site in the right MCS; this excises lacZalpha, giving blue/white selection in appropriate E.coli hosts. Cloned genes can be expressed from the tet promoter (inducible by tetracycline). The plasmid has oriT and can be mobilised.

pUS212

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E.coli cloning and expression vector. Has cloning sites downstream of Tet promoter. Clones genes of interest as SpeI-PstI fragments.

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