Principle approach and fusion cloning strategies

Copyright information:

Taken from "Translational control and target recognition by small RNAs "

Nucleic Acids Research 2007;35(3):1018-1037.

Published online 30 Jan 2007

PMCID:PMC1807950.

© 2007 The Author(s).

() Putative sRNA target sequences are cloned as translational fusions to on a low-copy vector that carries a pSC101* origin of replication (3–4 plasmid copies/cell) and confers chloramphenicol resistance. The fusion is transcribed from a constitutive λ P promoter (P derivative). Regulatory sRNAs are cloned on a high-copy vector that carries a ColE1 origin of replication (∼70 copies/cell) and confers ampicillin resistance. The sRNA gene is cloned under control of the constitutive P promoter (another derivative of λ P) such that transcription will precisely start at the native +1 site of the sRNA. For cells that carry both plasmids, the effect of a given sRNA on a target fusion can be determined by monitoring GFP fluorescence of colonies grown on agar plates, of liquid cultures grown in standard laboratory flasks or in microtiter plates, or by flow cytometry. Combinations of fusion and sRNA expression plasmids with control plasmids are used to determine (i) the basal fluorescence of cells and how it is affected by sRNA overexpression, (ii) the general effect of the plasmid-borne sRNA gene on GFP expression and (iii) the specific effect of an sRNA on a target fusion of interest. () Putative target sequences are PCR-amplified and cloned into specialized fusion vectors. If the target sequence is derived from a monocistronic gene or the first gene of an operon, and its promoter is known (left panel), it is amplified with an upstream primer that binds at the +1 site of the target gene and adds a BfrBI site, and a downstream primer that binds in the N-terminal region of the target gene and adds an NheI site in frame with the target gene coding region. The resulting PCR product is inserted into the standard fusion vector, pXG-10, digested with BfrBI/NheI. If the promoter +1 site is unknown (middle panel), the target sequence is amplified from cDNA of total RNA that was ligated to a 5′ RNA linker oligo upon treatment with TAP (this enzyme converts the 5′ PPP group of primary transcripts to 5′ P and thus allows the differential amplification of cDNAs that correspond to the native +1 site of an mRNA). The amplified cDNA will carry a 5′ BseRI site (contained in the RNA linker sequence). Insertion of the NheI/BseRI-digested cDNA into NheI/BsgI-digested RACE fusion plasmid, pXG-20, ensures that transcription of the fusion mRNA starts at the native +1 site of the target gene. Target sequences that are derived from within polycistronic mRNAs are amplified and cloned into the operon fusion vector pXG-30 (right panel). The upstream primer adds a BfrBI site in frame with the C-terminus of the upstream ORF; cloning into pXG-30 will create a C-terminal fusion to a short artificial reading frame composed of a FLAG epitope and a truncated gene, thus mimicking operon mRNA expression. See text and for more details.