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.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.