PCR reactions were performed with Q5 high-fidelity polymerase (New England BioLabs, Ipswich, MA, USA) using manufacturer instructions. These primers contain restriction sites for the enzymes AgeI and XmaI. coli K12 MG1655 by PCR with the primers 5′-tctcatACCGGTacgcccgcc-3′ and 5′-ctatCCCGGGtcaggctgttaccaaagaag-3′. Later, the transcription factor gene ( fucR) was obtained from the genome of E. The resulting plasmids were named pFUC_sfGFP_colE1 and pFUC_sfGFP_SC101.Ĭloning of FucR. Both DNA fragments were digested with the PstI-HF and EcoRI-HF restriction enzymes for 1 h at 37 ☌, gel purified, and ligated with T4 DNA ligase at room temperature for 1 h (New England Biolabs, Inc., Ipswich, MA, USA). coli K12 MG1655 genome, specifically from the fucose fucPIK operon. (IDT), including the PstI and EcoRI restriction sites at the 5′ and 3′ ends, respectively. The fucose-induced promoter ( pFuc) was synthesized as a gBlock from Integrated DNA Technologies, Inc. These plasmids were a kind donation from Dr. The high copy plasmid contains an ampicillin resistance gene as a selection marker and superfolder GFP (sfGFP) as a reporter molecule, while the low copy plasmid contains a chloramphenicol resistance and sfGFP controlled by pTac. Fucose biosensors were created in a high copy plasmid backbone (pTAC_sfGFP ColE1) and a low copy plasmid backbone (pTAC_sfGFP SC101). The in-silico plasmid construction was carried out in the SnapGene program, obtaining all the constructs and the primers ( Table 1). A biological method for quantifying fucose could be useful for nutraceutical and microbiological applications, as well as molecular diagnostics. In conclusion, a bacterial biosensor of fucose was validated with good sensitivity and precision. Finally, the biosensor was tested on different concentrations of free fucose and the supernatant of Bifidobacterium bifidum JCM 1254 supplemented with 2-fucosyl lactose, indicating the applicability of the method in detecting free fucose. Adjusting data to the Hill equation suggested non-cooperative, simple regulation of FucR to its promoter. The molecular circuit was specific against other monosaccharides and showed a linear response in the 0–45 mM range. coli BL21 transformed with a high copy plasmid containing pFuc and fucR displayed a high resolution across increasing fucose concentrations and high fluorescence/OD values after 18 h. For this, low- and high-copy plasmids were evaluated with and without the transcription factor fucR and its respective fucose-inducible promoter controlling the reporter gene sfGFP. Here we designed and developed a molecular quantification method of free fucose using fluorescent Escherichia coli. Methods for assessing fucose concentrations in complex media are lacking. Fucose metabolism is important for the production of short-chain fatty acids and is involved in cross-feeding microbial interactions. The bacterial consumption of fucose and fucosylated HMOs is critical in the gut microbiome assembly of infants, dominated by Bifidobacterium. In humans, fucose can be found in human milk oligosaccharides (HMOs), mucins, and glycoproteins in the intestinal epithelium. Prices start at $295.00 per year and up.L-Fucose is a monosaccharide abundant in mammalian glycoconjugates. Not all features available in SnapGene Viewer. Freely share data with your colleagues or customers using the universally accessible SnapGene format.ģ0-Day Trial.Export a plasmid map as an image, or export an annotated DNA sequence to GenBank format.Identify open reading frames (ORFs) with a mouse click.Design and annotate primers for PCR, sequencing, or mutagenesis.Automatically annotate common features or manually annotate coding sequences and other features.Search a DNA sequence to match either a DNA query, a protein translation, or an annotation.Browse or print a DNA sequence and its annotations using customizable Map, Sequence, Enzymes, Features, Primers, and History views.Create a DNA sequence file by either entering a sequence, importing a record from GenBank, or opening an annotated sequence stored in one of many common file formats.SnapGene Viewer allows molecular biologists to create, browse, and share richly annotated DNA sequence files up to 1GB in length.
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