Various hfq RBS sequences and plasmid construction
This protocol is extracted from research article:
Optimized expression of Hfq protein increases Escherichia coli growth
J Biol Eng, Feb 18, 2021; DOI: 10.1186/s13036-021-00260-x

Different E. coli hfq variants with a variety of translation efficiencies were designed by RBSDesigner. The variants 1–4 were designed by RBSDesigner. To achieve a higher expression level, randomly generated RBS sequences were screened, and variants 5 and 6 were selected. Sequences of primers and genes are shown in Table S2.

Briefly, the amplified hfq gene from E. coli DH5α strain using HfqF_AatII and HfqR_XhoI primers was cloned into the corresponding restriction sites in pSC101 plasmid. A strong transcriptional terminator T1/TE was cloned downstream of the inserted hfq sequence. The hfq variant plasmids were constructed by inverse PCR on the pSC-WT hfq template. pSC-hfq-x-gfp had the gfp gene cloned downstream of the Hfq coding sequence using SpeI/XhoI and thereby their coding sequences were fused. To increase the structural flexibility between Hfq and GFP proteins, a stretch of Gly and Ser residues (“GS” linker) was used to connect hfq and gfp genes. All constructed RBS sequences of the hfq variants are listed in Table S3.

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