. Author manuscript; available in PMC: 2023 Jun 5.

Published in final edited form as: Nat Protoc. 2022 Feb 23;17(4):941–961. doi: 10.1038/s41596-021-00657-4

Table 1.

Plasmids for use in the B3H assay or for use as vectors for cloning new plasmids with Baits and Preys of interest.

Plasmid Type	Plasmid Info	Expresses	Description	Name	Details	Ref
pAdapter	pAC; p15A origin; CmR	λCI-MS2^CP	Encodes λCI fused via 12aa linker (AAAEFPGIHPGM) to a multimerization-resistant MS2^CP; transcription driven by various promoters.	pKB989	Under control of lacUV5 promoter	¹²
				pCW17 (addgene #: 174664)	Under control of a constitutive promoter	¹⁶
				p35u4 (addgene #: 174786)	Under control of a constitutive promoter	¹⁵
		λCI-empty	Negative control plasmid lacking MS2^CP (encodes λCI alone)	pAC-λCI (addgene #: 53730)	corresponds to pKB989	¹³
				pRM16 (addgene #: 174787)	corresponds to pCW17
				pRM17 (addgene #: 174788)	corresponds to p35u4
pPrey	pBR322 origin; CarbR	α-Hfq	Encodes residues 1–248 of α fused via three alanine residues to prey protein, under the control of tandem lpp and lacUV5 promoters; Cloning sites: NotI + BamHI	pKB817 (addgene #174661)	.Prey: full-length wild-type Ec Hfq	¹²
		α-ProQ		pKB949	Prey: full-length wild-type Ec ProQ	¹⁶
		α-empty	Encodes full-length rpoA (α), under the control of tandem lpp and lacUV5 promoters; should not be used for cloning, as it lacks restriction sites.	pBR-α (addgene #53731)	Negative control plasmid lacking prey protein	¹³
pBait	pCDF; CloDF13 origin; SpecR;	1XMS2^hp- ChiX	Encodes a single MS2 hairpin sequence upstream of Bait RNA sequence, under the control of an arabinose inducible promoter; Cloning sites: XmaI + HindIII	pCH6 (addgene #174662)	RNA Bait: E. coli chiX	¹⁶
		1XMS2^hp- cspE 3’UTR		pSP10	RNA Bait: E. coli 3’UTR of cspE	¹⁶
		1XMS2^hp- SibB		pSP14	RNA Bait: E. coli sibB.	¹⁶
		1XMS2^hp- OxyS		pCH9	RNA Bait: E. coli oxyS.	¹⁶
		1xMS2^hp -empty		pCH1 (addgene #174663)	A 14bp multiple cloning site is present between XmaI and HindIII sites.	¹⁶