Table S2. CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78C101%. Conclusions The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the CC-671 detection of fipronil or nanobody-based assays in general. and are both in the operon of MSR-1, and have a minor and nonessential function in magnetite biomineralization [11]. Thus, MamC and MamF have been frequently exploited for the magnetosomal display of functional proteins such as immunoglobulin G-binding domains [12], thyroid-stimulating hormone receptor (TSHR) [13] and Staphylococcal protein A (SPA) [14]. The variable domain of Camelidae heavy-chain-only antibodies (VHHs), also referred to as nanobodies (Nbs), have superior properties to conventional antibodies for their small size, non-immunogenicity, thermal stability, high solubility, ease of production in microorganisms and Rabbit polyclonal to OLFM2 ease of genetic modification [15, 16]. The extensive availability of molecular biological techniques facilitates the genetic conjugation of Nbs to magnetosomes, but only one investigation on the fusion expression of Nbs binding red fluorescent protein (RFP) on magnetosomes has been reported so far [17]. Over the past few years, Nbs are attractive in the field of immunoassays for monitoring small molecules such as biomarkers and environmental pollutants [18]. Nbs chemically conjugated to magnetosomes proved to be an effective tool for the detection of environmental compounds [19, 20]. It is promising to display nanobodies on magnetosomes by a genetic method to monitor small-molecule contaminants. In our previous CC-671 study, Nbs selective to fipronil and its metabolites were generated and used for the detection of fipronil in rodent sera [21]. Fipronil is the first phenylpyrazole insecticide that acts at the -aminobutyric acid (GABA) receptor of insects, blocking the passage of chloride ions [22]. It is widely used for the control of field insects in agriculture as well as fleas and ticks on pets [23]. Nevertheless, the widespread use of fipronil CC-671 has induced an increasing concern about the possible off-target harm to ecosystems and human health particularly when employed in non approved application [24]. Here, we displayed a Nb (F1) on magnetosomes by gene fusion using MamC and MamF as anchoring proteins in MSR-1. MSR-1 gives the highest magnetosome yields among the MTB [25], to construct functional MNPs specific for fipronil. The modified magnetosomes could be easily produced with low cost, having a potential application in the field of immunoassays. Methods and materials Bacteria strains and culture conditions The bacteria strains and plasmids used in this study are listed in Additional file 1: Table S1. strains were cultured in CC-671 Luria broth (LB) at 37?C. MSR-1 was cultured in sodium lactate medium (SLM) or sodium glutamate medium (SGM) (substitute 4?g sodium glutamate for NH4Cl and yeast extract in SLM) as described previously [26]. Large-scale MSR-1 cells were fed-batch cultured in CC-671 a 7.5 L-fermenter according to the method reported by Zhang et al. [27]. Antibiotics prepared in culture media were as follows: for and upstream region, downstream region, and uFFd, a cassette consisting of upstream region, downstream region, were assembled by fusing PCR amplification. uCFd and uFFd were then subcloned into pK18mobSacB digested with I and I to construct the suicide recombinant plasmids pKCF and pKFF, respectively. CF, a fusion gene consisting of and Nb gene, was amplified from uCFd with primers MamC-F (I) and Fip-R (I), and subcloned into pBBR1MCS-2 to create pBBRCF. pKCF was transferred to wild type (WT) MSR-1 to generate a double crossover strain CF, which was successively cultured in medium containing kanamycin and 10% sucrose. pKFF and pBBRCF were then transferred to the strain CF to generate CFFF and CF?+?, respectively. Strains CF?+?and CFFF were cultured in medium containing kanamycin. Extraction of Nb-magnetosomes Cells were harvested by centrifugation (8000encodes levansucrase coverting sucrose to levans, lethal to many Gram-negative bacteria (e.g., RS-1) [30], it is commonly used as a counterselection marker. A.