19 research outputs found

    Toward Single Electron Nanoelectronics Using Self-Assembled DNA Structure

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    DNA based structures offer an adaptable and robust way to develop customized nanostructures for various purposes in bionanotechnology. One main aim in this field is to develop a DNA nanobreadboard for a controllable attachment of nanoparticles or biomolecules to form specific nanoelectronic devices. Here we conjugate three gold nanoparticles on a defined size TX-tile assembly into a linear pattern to form nanometer scale isolated islands that could be utilized in a room temperature single electron transistor. To demonstrate this, conjugated structures were trapped using dielectrophoresis for current–voltage characterization. After trapping only high resistance behavior was observed. However, after extending the islands by chemical growth of gold, several structures exhibited Coulomb blockade behavior from 4.2 K up to room temperature, which gives a good indication that self-assembled DNA structures could be used for nanoelectronic patterning and single electron devices

    Specificity of core-bradavidin binding to Brad-tag analyzed by biolayer interferometry.

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    <p>(<b>A</b>) Anti-Penta-HIS biosensors were functionalized with Brad-tag–EGFP–His-tag fusion protein (step 1, arrow in the graph). After a brief wash (10 s) in measurement buffer, biosensors were incubated with a series of different avidin proteins at concentration of 0.06 mg/ml (step 2). Sample without any avidin protein was used as a negative control (buffer). Finally, biosensors were exposed to the measurement buffer leading to dissociation of the bound core-bradavidin proteins (step 3). (<b>B</b>) The measured raw data for buffer is subtracted from the raw data of different proteins.</p

    Stick model (stereo view) of the ligand-binding site of wt bradavidin.

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    <p>(<b>A</b>) The carbon atoms of the Brad-tag sequence (subunit I) are shown in blue and the residues around the Brad-tag sequence in magenta (subunit III) and yellow (subunit IV). Structural water molecules near the Brad-tag sequence are shown as red spheres. (<b>B</b>) Superimpositioning of the ligand-binding site of wt bradavidin and chicken avidin [PDB: 1AVD]. Colouring for wt bradavidin as in (A); the carbon atoms of residues of chicken avidin are shown in grey. The residues Asn12, Leu14, Ser16, Tyr33, Trp70, Ser73, Ser75, Thr77, Phe79, Trp97, Leu99 and Asn118 of chicken avidin were superimposed to the equivalent residues Asn9, Tyr11, Ser13, Tyr31, Phe66, Cys69, Ser71, Thr73, Trp75, Trp89, Leu91 and Asp107 of wt bradavidin. For clarity, the Brad-tag sequence is not shown. For chicken avidin, the residue numbers are shown in brackets. BTN, biotin; *, Tyr31 (Tyr33); **, Asn33 (Thr35).</p

    Four tyrosine residues at the intersection of all four subunit interfaces in bradavidin.

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    <p>A stereo view. The tyrosine residues are shown as stick models with different colouring for the different subunits (subunit I, blue; II, cyan; III, magenta; and IV, yellow). Structural water molecules are shown as red spheres. Electron density map (a weighted 2FO-FC map; sigma level 1) around the water molecules and the side chain oxygen atoms of Tyr90 is shown in blue. Putative hydrogen bonds are indicated with grey dashes.</p

    X-ray structure determination statistics for wt bradavidin [PDB: 2Y32].

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    a<p>The numbers in parenthesis refer to the highest resolution bin.</p>b<p>From XDS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035962#pone.0035962-Kabsch1" target="_blank">[53]</a>.</p>c<p>From Refmac 5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035962#pone.0035962-Murshudov1" target="_blank">[57]</a> using TLS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035962#pone.0035962-Winn1" target="_blank">[71]</a> & restrained refinement.</p

    Purification of EGFP fusion protein using N-terminal Brad-tag.

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    <p>(<b>A</b>) Photograph of Brad-tag–EGFP–His-tag bound to core-bradavidin resin under UV-light. First, core-bradavidin was coupled by amine groups to the terminal NHS carboxylates of the linkers (resin–NH–(CH<sub>2</sub>)<sub>5</sub>–COONHS). Then, Brad-tag–EGFP–His-tag (prepurified with Ni-NTA column) was incubated with the functionalized resin and the resin was pelleted. In the absence (–) of biotin Brad-tag–EGFP–His-tag concentrates on the resin pellet. The presence (+) of free biotin inhibits the binding. Stoichiometry and the size of compounds in the schematic figure are only speculative. (<b>B</b>) SDS-PAGE analysis of the protein purification experiment for cellular lysates of N-Brad-tag–EGFP-C. Cleared cellular lysate (total) was incubated with core-bradavidin resin. Then the resin was washed with buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.5) and samples 1 to 5 were eluted. Molecular weight markers (M, kDa) are indicated on the left. (<b>C</b>) The fluorescence spectra measured for cleared cellular lysate (total) and eluted samples 1 to 5 from the protein purification experiment for Brad-tag–EGFP.</p

    Structure comparison of wt bradavidin, wt streptavidin [PDB: 2BC3] and chicken avidin [PDB: 1AVD].

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    <p>(<b>A</b>) Cartoon models of tetrameric proteins. Subunits are shown in different colors as follows: I (blue), II (cyan), III (magenta) and IV (yellow). The key biotin-binding pocket occupying residues (wt bradavidin and wt streptavidin) and biotin (chicken avidin) are shown as spheres. The N- and C-termini are indicated by letters. (<b>B</b>) Superimposition of the Cα traces of subunits I of wt bradavidin (blue), wt streptavidin (orange) and chicken avidin (cyan). The Cα trace for C-terminal residues starting from Lys127 of subunit III of wt bradavidin is shown, too. The ligand-binding site occupying residues Glu131-Leu133 of wt bradavidin and Gly151-Pro153 of wt streptavidin, and the biotin ligand of avidin, are shown as sticks. The left and right arrows pinpoint the N- and C-terminal sites, respectively, were major differences are seen between the proteins. Trp5 (left arrow) of wt bradavidin is shown as sticks. The N- and C-termini are indicated and loops are numbered. (<b>C</b>) Monomeric cartoon models. The N-terminus and C-terminus of each protein are indicated in red and blue, respectively, the colouring starting from equal positions in all proteins. The key residues occupying the biotin-binding site are shown as sticks. (<b>D</b>) Loop design. Colouring of subunits are as in (A) and representation of the key residues as in (C). The arrow pinpoints the varying beginnings of the L7,8-loops in the three structures. The L3,4-loop of the wt streptavidin structure is not fully visible (dashed line).</p

    Microwave hydrolysis, as a sustainable approach in the processing of seaweed for protein and nanocellulose management

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    The nature of marine biomass is very complex for a material scientist due to the large seasonal variation in the chemical composition that makes it difficult to prepare standardized products. A systematic investigation of the interaction of microwave irradiation with seaweed from Norway and Caribbean region was performed, covering a broad temperature range (130 → 170 ◩C) and without and with addition of â„œ-valerolactone (GVL) in ratios of 1:4 and 1:2. The temperatures above 150 ◩C and without addition of GVL led to the closure of mass balances up to 90 % that includes polysaccharides, “pseudo-lignin” fraction, fatty acids, and proteins. Fucoidan and mannose represented >50 % of all detected polysaccharides in ascophyllum nodosum (AN), while aegagropila linnaei (AL) contained mostly glucose. The presence of arabinose and rhamnose in the upper surface of the cell wall hinders the glucose release during microwave treatment. The differences in the polysaccharide composition among both algae samples hindered the definition of a parameters set that can be used in microwave treatment of various seaweed species. A large fraction of protein (> 95 %) remained in the seaweed solid residue. Higher amount of protein was determined in AL, which was dominated by leucine and lysine. Another potential barrier to the application of seaweed in industry is the limited knowledge on the chemical composition of “pseudo-lignin” and extractives. The total amino acid analysis was identified as the most accurate to characterize the protein yield and composition. The results showed that microwave treatment of seaweed is indeed a viable method for producing bioactives in the temperature range 120–150 ◩C, and proteins and nanocellulose at temperatures above 170 ◩C without using GVL. The microwave temperature and seaweed type played a dominating role in the mass closure balances leading to >95 % identified compound.</p

    Brad-tag–EGFP fusion proteins.

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    <p>(<b>A</b>) Four different Brad-tag–EGFP fusion protein constructs were used in the current study. Brad-tag was positioned at the N- or C-terminus of the fusion proteins. A His-tag was also included in two of the constructs directly after the sequence of EGFP. (<b>B</b>) Immunoblot analysis using antibody against GFP was used to evaluate the quality and amount of tagged EGFPs. Biotinylated–EGFP was used as a positive control (Vikholm-Lundin et al, unpublished) and core-bradavidin as a negative control. Numbers in brackets indicate different protein productions. Molecular weight markers (M, kDa) are indicated on the left. (<b>C</b>) The fluorescence spectra measured for purified Brad-tag–EGFP–His-tag (12.4 ng/”l) and clarified cellular lysates of other Brad-tag–EGFP constructs.</p
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