Chrna2-cre transgenic C57BL6 mice were generated by introducing the Cre gene at the ATG site of the first coding exon (middle of exon2) of the Chrna2 gene in a Bacterial Artificial Chromosome (BAC, RP23–48P22). The protocols used and information of the bacterial strain (EL250) are available on http://recombineering.ncifcrf.gov . A plasmid containing nls-Cre-SV40 polyA-FRT-Kan/Neo-FRT was generated as a PCR template (information on request). Successful introduction of the Cre construct into the BAC was confirmed with PCR using the primers GACAGCCATTTTCTCGCTTC and AGGCAAATTTTGGTGTACG in a standard PCR reaction, the same primers were subsequently used for genotyping of the mice. BAC plasmid length was analyzed by enzyme restriction (2 h, 37°C), followed by Pulse Field Gel Electrophoresis (PFGE, CHEF mapper, Bio-Rad) 6V/h, 18h, 120° switch, 1–20s switch time. Validation sequencing of the BAC construct was made with custom designed primers covering the modified region (MWG-Biotech AG, Ebersberg, Germany). The modified BAC was linearized by NotI and purified as described in Marshall et al48 (link). Briefly, the BAC DNA was separated with PFGE followed by β-agarase (NEB) digestion and dialysis to exchange the buffer to injection buffer. The modified BAC, which includes ~100 kbp upstream and ~8 kbp downstream of the Chrna2 gene as well as all introns, was linearized by cleaving with NotI and introduced randomly into the mouse genome by pronuclear injection at Uppsala University Transgenic Facility (UUTF), which resulted in a founder line with expression of Cre in cells expressing CHRNA2 protein. Viaatlx mice22 (link) and Gt(ROSA)26Sortm14(CAG-tdTomato)Hze (R26tom; Allen Brain Institute) mice have been described elsewhere. All animal procedures were approved by the appropriate local Swedish ethical committee (Jordbruksverket). Efforts were made to minimize the numbers of animals used.
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Agarase
Agarase
Agarase is an enzyme that catalyzes the hydrolysis of agarose, a complex polysaccharide derived from certain red algae.
This enzyme plays a crucial role in the degradation of agar, a gelatin-like substance used widely in microbiology and biotechnology.
Agarase is produced by various marine bacteria and fungi, and it has applications in the extraction and purification of nucleic acids, as well as in the development of novel biomaterials.
Reasearch on agarase is important for understanding its sructure, function, and potential utilization in diverse fields such as food processing, pharmaceuticals, and environmental remediation.
The PubCompare.ai tool can enhace the reproducibility of agarase research by helping scientists quickly identify the best experimental protocols from the literature, preprints, and patents using advanced AI analysis.
This enzyme plays a crucial role in the degradation of agar, a gelatin-like substance used widely in microbiology and biotechnology.
Agarase is produced by various marine bacteria and fungi, and it has applications in the extraction and purification of nucleic acids, as well as in the development of novel biomaterials.
Reasearch on agarase is important for understanding its sructure, function, and potential utilization in diverse fields such as food processing, pharmaceuticals, and environmental remediation.
The PubCompare.ai tool can enhace the reproducibility of agarase research by helping scientists quickly identify the best experimental protocols from the literature, preprints, and patents using advanced AI analysis.
Most cited protocols related to «Agarase»
agarase
Animals
Animals, Transgenic
Bacteria
Bacterial Artificial Chromosomes
Brain
Buffers
Cells
Dialysis
Digestion
DNA Restriction Enzymes
Electrophoresis
Electrophoresis, Gel, Pulsed-Field
Exons
Genes
Genome
Introns
Mice, Laboratory
Mice, Transgenic
Oligonucleotide Primers
Plasmids
Poly A
Proteins
Pulse Rate
Rosa
Simian virus 40
Strains
tdTomato
The previously assembled single-pot39 (link) and immune library3 (link) were stored as both phage and glycerol stocks, the latter containing enough representations to seed 6 x 400 mL cultures to an OD of 0.1 for re-rescue, ie. approx. 1.2 e+11 cfu. One 2 mL glycerol was made to 24 mL with media and divided into 12 x 2 mL and miniprepped (Qiagen) for elution of approx. 12 x 10 µg in 80 µL of EB. The destination vectors pecan126, pecan133 and pecan 164 were modified by replacement of the MBG B sdAb with a 2 kbp tet stuffer from pecan 21 via NcoI (partial)/ NotI to provide a convenient marker for scoring positive sdAb gene inserts and to enable SfiI/SfiI cloning21 (link) for the single-pot retrofit while immune libraries employed SfiI/NotI. Each vector was grown in 12 x 3.5 mL of terrific broth, 2% glucose and appropriate antibiotics and 12 x 2 mL miniprepped for elution of approx. 12 x 10 µg in 80 µL EB which were then pooled. Vectors and inserts were digested by addition of 120 µl 10 x React2 buffer, 120 µL 10 x BSA and 24 µL of SfiI (20 U/µL) and left for 12–18 h in a 50°C oven. The immune library inserts and vectors were further digested by addition of 60 µL React 3 and 24 µL NotI (10 U/µL) for a further 12 h at 37°C. Vector digests were CIPped by addition of 120 µL 10xCIP buffer and 40 µL CIP (1 U/µL) and left at 37°C for 2 h (although self-ligation is not an issue here, we dephosphorylate to reduce inter vector-vector ligation reducing the available destination DNA for the insert). DNA was electrophoresed on 1% (for insert) or 0.5% (for vector) GTG TAE gels and the appropriate bands excised in a volume of about 2.5–3.5 mL for a scaled-up version of in agarose ligation56 . The gels were melted at 70°C then kept at 37°C and both vector and insert were added and mixed in 400 µL aliquots into 6–8 tubes each containing 780 µL water, 200 µL 10xT4 DNA ligase buffer (Promega) and 20 µL β-Agarase (1U/µL) which stayed liquid at room temperature (approx. 75F). 20 µL of T4 DNA ligase (1 U/µL) was added, the tubes mixed gently by inversion, covered in foil and left at slightly warmer than room temperature atop the hybridization oven for 18–24 h. The ligations were aliquoted to 24–32 x 500 µL, each extracted with 450 µL of phenol/chloroform (Invitrogen), and 450 µL supernatants precipitated by addition of sodium acetate/ethanol and left on the bench for 2 h only to avoid excessive salt precipitation. Tubes were microfuged at 13.5 krpm for 15 min, supernatants poured off, pellets briefly washed with 350 µL 70% EtOH, recentrifuged for 5 min, aspirated and dried briefly in a tissue culture hood. Each pellet was resuspended in 42–32 µL (depending on the number of ligations) and pooled to provide a combined 32 aliquots of 60 µL for bulk electroporation which were aliquoted for storage at −80°C until required.
HBV88 or HB2151+pecan134 were made electrocompetent using low temperature growth57 (link) in low salt YENB media58 (link) growth followed by extensive washing59 (link) and reliably yielded transformation efficiencies in the mid 1e+9 cfu/µg ccc pUC19 range only some 2–4 fold less than home-made preparations of high efficiency strains such as DH10B or DH10F'tet. A streak on M9/chloramphenicol + thiamine minimal agar to select the F'-episome was rinsed into 400 mL of liquid M9 equivalent and shaken overnight at 37°C. From this starter, 6x400 mL flasks of YENB plus chloramphenicol were inoculated to an OD600 nm of 0.05 cm−1 and shaken at 25°C until an OD of between 0.4 and 0.5 was reached (approx. 6–7 h). Cells were immediately pelleted (Allegra GPR, 4x750 mL pots, swing out rotor, 4°C, 20 min), resuspended gently in a total volume of approx. 1.5 L ice cold water and re-pelleted (Allegra, 6x250 mL, fixed angle, 5.75 krpm, 20 min 4°C). Cells were washed in water once more, then washed in 15% glycerol and finally resuspended in 8.7 mL of 15% glycerol and aliquoted to 32 x 270 µL, snap frozen in −80°C isopropanol and stored frozen until required.
Electroporations of the 60 µL ligation and 270 µL cell aliquots were performed using an electroporator (BioRad) at 2.5 kV with 2 mm gap electrocuvettes (Bulldog Bio Inc., Portsmouth, NH) that accommodated the combined volume. Following electroporation the cuvette contents were poured into 50 mL Falcon tubes followed by 3 cuvette washes of 2 mL of prewarmed (37°C) SOB 2% glucose with two electroporations worth pooled in each Falcon tube. The mixes were left static for 60 min and cells were then pelleted (Allegra GPR, 3 krpm, 10 min, 20°C), resuspended in 400 µL supernatant and spread on Bioassay dishes containing 250 mL 2xTY 2% glucose with appropriate antibiotics. Following growth overnight, cells were scraped with a 3” wallpaper scraper (Hyde, Home Depot, San Antonio) to a pot and the plates also washed with 4 mL of 2xTY 2% glucose which was added to the pot and the whole lot mixed thoroughly. After measuring the OD600nm*, 6 flasks of 400 mL 2xTY 2% glucose were seeded to and OD600nm of 0.05, grown with shaking at 37°C to an OD of 0.4–0.5 and infected with M13K07 at an moi of 20 for 1 h static. Kanamycin, IPTG and arabinose was added according to the system employed and rescue proceeded for 18–24 h at 30°C. Cultures were clarified by centrifugation (Sorvall RC6+, 4x1 L, 8 krpm, 1 h, 4°C), pooled and precipitated by addition of 480 mL of 20% PEG6000/2.5 M NaCl and overnight stirring at 4°C. Phagemids were pelleted (Allegra GPR, 3.75 krpm, 4x750 mL swing out, 1 h, 4°C), drained and resuspended in a total volume of 16 mL PBS to which was added 16 mL of glycerol, and 16 x 2 mL aliquots stored at −80°C until required. *The remainder of the cell suspension was combined with an equal volume of ice cold 30% glycerol in terrific broth and aliquoted into 40–50 2 mL cryovials such that each aliquot could seed another six flasks if required.
HBV88 or HB2151+pecan134 were made electrocompetent using low temperature growth57 (link) in low salt YENB media58 (link) growth followed by extensive washing59 (link) and reliably yielded transformation efficiencies in the mid 1e+9 cfu/µg ccc pUC19 range only some 2–4 fold less than home-made preparations of high efficiency strains such as DH10B or DH10F'tet. A streak on M9/chloramphenicol + thiamine minimal agar to select the F'-episome was rinsed into 400 mL of liquid M9 equivalent and shaken overnight at 37°C. From this starter, 6x400 mL flasks of YENB plus chloramphenicol were inoculated to an OD600 nm of 0.05 cm−1 and shaken at 25°C until an OD of between 0.4 and 0.5 was reached (approx. 6–7 h). Cells were immediately pelleted (Allegra GPR, 4x750 mL pots, swing out rotor, 4°C, 20 min), resuspended gently in a total volume of approx. 1.5 L ice cold water and re-pelleted (Allegra, 6x250 mL, fixed angle, 5.75 krpm, 20 min 4°C). Cells were washed in water once more, then washed in 15% glycerol and finally resuspended in 8.7 mL of 15% glycerol and aliquoted to 32 x 270 µL, snap frozen in −80°C isopropanol and stored frozen until required.
Electroporations of the 60 µL ligation and 270 µL cell aliquots were performed using an electroporator (BioRad) at 2.5 kV with 2 mm gap electrocuvettes (Bulldog Bio Inc., Portsmouth, NH) that accommodated the combined volume. Following electroporation the cuvette contents were poured into 50 mL Falcon tubes followed by 3 cuvette washes of 2 mL of prewarmed (37°C) SOB 2% glucose with two electroporations worth pooled in each Falcon tube. The mixes were left static for 60 min and cells were then pelleted (Allegra GPR, 3 krpm, 10 min, 20°C), resuspended in 400 µL supernatant and spread on Bioassay dishes containing 250 mL 2xTY 2% glucose with appropriate antibiotics. Following growth overnight, cells were scraped with a 3” wallpaper scraper (Hyde, Home Depot, San Antonio) to a pot and the plates also washed with 4 mL of 2xTY 2% glucose which was added to the pot and the whole lot mixed thoroughly. After measuring the OD600nm*, 6 flasks of 400 mL 2xTY 2% glucose were seeded to and OD600nm of 0.05, grown with shaking at 37°C to an OD of 0.4–0.5 and infected with M13K07 at an moi of 20 for 1 h static. Kanamycin, IPTG and arabinose was added according to the system employed and rescue proceeded for 18–24 h at 30°C. Cultures were clarified by centrifugation (Sorvall RC6+, 4x1 L, 8 krpm, 1 h, 4°C), pooled and precipitated by addition of 480 mL of 20% PEG6000/2.5 M NaCl and overnight stirring at 4°C. Phagemids were pelleted (Allegra GPR, 3.75 krpm, 4x750 mL swing out, 1 h, 4°C), drained and resuspended in a total volume of 16 mL PBS to which was added 16 mL of glycerol, and 16 x 2 mL aliquots stored at −80°C until required. *The remainder of the cell suspension was combined with an equal volume of ice cold 30% glycerol in terrific broth and aliquoted into 40–50 2 mL cryovials such that each aliquot could seed another six flasks if required.
1% agar (catalog number A1296, Sigma-Aldrich Corp., St. Louis, MO, USA) containing 0.2% carboxymethylcellulose (catalog number C4888, Sigma-Aldrich Corp., St. Louis, MO, USA) was poured into petri dishes or into the wells of a 96-well plate, respectively. Stock solutions of polymer-degrading enzymes were dissolved in 1× Tris-buffered saline (TBS) buffer or deionized water, following the suppliers’ instructions for reconstitution of the enzyme. Spot plating was performed with 5 μL of commercial cellulase from Aspergillus niger (catalog number C1184, Sigma-Aldrich Corp., St. Louis, MO, USA) at different concentrations (0.1 and 5 μg/μL). Agarase (1 μg/μL) (catalog number EO0461, Fermentas, Burlington, ON, Canada), amylase (1 μg/μL) (catalog number 10065, Sigma-Aldrich Corp., St. Louis, MO, USA) and proteinase K (1 μg/μL) (catalog number EO0491, Fermentas, Burlington, ON, Canada) were used as control enzymes. In some cases, agar was replaced by 1% agarose or 0.75% Gelrite with or without 0.2% CMC, respectively. All plates were incubated at 27 °C for 12–16 h after which hydrolysis zones were visualized by flooding of the plates/wells with Gram’s iodine (2 g potassium iodide and 1 g iodine in 300 mL water) for 5 min followed by a rinses with deionized water. For detection with Congo Red, the plates were flooded with 0.1% Congo Red for 15–20 min and then rinsed with 1 M NaCl. Plates where CMC was omitted were used as non-substrate controls in all experiments.
Agar
agarase
Amylase
Aspergillus niger
Carboxymethylcellulose
Cellulase
Endopeptidase K
Enzymes
Gelrite
Hydrolysis
Hyperostosis, Diffuse Idiopathic Skeletal
Iodine
Polymers
Potassium Iodide
Saline Solution
Sepharose
Sodium Chloride
agarase
Anti-Antibodies
Cells
Digestion
Microscopy
Pulse Rate
Replicon
Sepharose
Asynchronous exponentially growing cells were labeled with 20 μmol/L IdU for 20 min. They were then washed in phosphate-buffered saline (PBS), and subsequently labeled with 50 μmol/L CldU for 20 min, then cells were chased with of 200 μmol/L of thymidine for 60-90 min. Cells were trypsinized and resuspended in PBS. Cells were then embedded in pulsed-field gel electrophoresis agarose plugs to prepare high-molecular-weight genomic DNA. After proteinase K digestion, agarose plugs were washed with TE buffer. Agarose plugs were then melted in 100 mmol/L MES (pH 6.5), and digested using 2 μL of β-agarase (Biolabs). The DNA solution was poured into a Teflon reservoir and DNA was combed onto silanized coverslips (Microsurfaces, Inc.) using a combing machine. Samples have included a BAC as a molecular marker, allowing us to convert labeled signal length (in microns) to fiber length (in kb) as molecular combing was previously shown to uniform stretching 32 (link). Uniform stretching was confirmed by the uniform length of the stretched marker DNA. Coverslips were visually examined for DNA density and fiber length by a preliminary staining with YoYo1 (invitrogen). Sample preparations containing fibers that were mostly shorter than 200 kb or DNA concentration was too low or too high were discarded. The average fiber length in our studies is about 400 kb. For slide preparations that contained long fibers with appropriate density, coverslips with combed DNA were incubated at 60 °C for 2 h and denatured in 0.5 N NaOH for 20 min. IdU was detected using a mouse antibody directed against BrdU (IgG1, Becton Dickinson, cat.347580, 1:25). CldU was detected using a rat antibody directed against BrdU (Accuratechmecal, cat. OBT0030, 1:50). Single strand DNA (ssDNA) was detected using a mouse antibody directed against ssDNA (IgG 2a, Chemicon, cat. MAB3034, 1:100). Coverslips with DNA were incubated with primary antibodies for 1 h at room temperature. After washes, samples were incubated with secondary antibodies for 45 min. Secondary antibodies included Alexa Fluor 594 donkey antibody directed against rat (A21209, 1:100), Alexa Fluor 488 goat antibody directed against mouse IgG1 (γ1) (A-21121, Invitrogen, 1:100), and Alexa Fluor 647 goat antibody directed against mouse IgG2a (γ2a) (A21241, Invitrogen, 1:100). Slides were scanned with a BD pathway 855 controlled by AttoVision (Becton Dickinson). Fluorescent signals were measured using ImageJ (from the National Cancer Institute) with custom-made modifications. Only replication signals from high quality ssDNA (not from DNA bundles, not located at the end of a strand) were selected for analyses. Signals were marked for evaluation by “blind” measurers (not knowing which samples they were measuring); signal length was measured using the Image J software followed by automatic compilation of signal lengths into an Excel worksheet. Fork velocities and origin distances were calculated using Excel with a constant of 2kb/μm. Replication fork velocities were calculated using elongating fork signals only (initiating forks were eliminated; see a recent review62 (link) for a discussion of good practices in dynamic molecular combing). Experiments were performed at least in duplicate using independent biological isolations of DNA fibers for each experimental condition. Statistical analysis was performed in Prism 5 or 6 (GraphPad software) using the non-parametric Mann-Whitney rank sum test. Normality of distributions was assessed using the Kolmogorov-Smirnov test.
Most recents protocols related to «Agarase»
A qualitative test for the agarase enzyme was performed with iodine using Lugol's staining process. Selected isolates were grown in ZMB (HiMedia, Kennett Square, PA, USA), pH 7.6 ± 0.2. An 8 mm well was cut in the agar, and 100 µL broth culture of each strain was added into the well and incubated at 30°C for 3 days. After incubation, the agarolytic activity was measured by adding 2 mL Lugol's iodine on the entire surface of the plate and leaving it for 15 min [36] (link). Additionally, the agarolytic index (AI) was calculated as the ratio between the diameter of the clear zone and the colony. This index denotes the ability of the bacteria to produce agarase enzymes [36] (link). The calculating formula is shown below: AI = Clear zone diameter (mm) -Colony diameter (mm)/Colony diameter (mm)
All experiments were conducted following the protocols outlined in the Sambrook and Russell manual.
Primers utilized in this study were synthesized and obtained from Invitrogen, USA. Escherichia coli (E. coli) DH5α served as the host strain for plasmid maintenance. Pseudomonas aeruginosa BC15 was employed as the host strain for housing the constructed plasmid containing the Cd sensing element and novel reporter gene.
Plasmid DNA was utilized to transform E. coli DH5α via a calcium chloride transformation method, while transformation in P. aeruginosa BC15 was performed through electroporation using a Gene Pulser (Bio-Rad, USA). Electroporation conditions included a 2.5 kV/cm eld strength, a 25 mF capacitor, and a 200 Ω resistor (Periasamy et al. 2015).
Primers utilized in this study were synthesized and obtained from Invitrogen, USA. Escherichia coli (E. coli) DH5α served as the host strain for plasmid maintenance. Pseudomonas aeruginosa BC15 was employed as the host strain for housing the constructed plasmid containing the Cd sensing element and novel reporter gene.
Plasmid DNA was utilized to transform E. coli DH5α via a calcium chloride transformation method, while transformation in P. aeruginosa BC15 was performed through electroporation using a Gene Pulser (Bio-Rad, USA). Electroporation conditions included a 2.5 kV/cm eld strength, a 25 mF capacitor, and a 200 Ω resistor (Periasamy et al. 2015).
Qualitative detection of Cd by CBOWCB. β-agarase is used as a reporter gene that cleaves agar into neoagarobiose with D-galactose reducing end. The cells harboring pUCP-P cadR -dagA (cadR gene promoter with β-agarase) were inoculated in LB broth supplemented with 0.3% agar at 37°C for 24 h with different concentrations of 20 to 100ppm of CdCl 2 . The culture broth was harvested by centrifugation at 14,000 rpm for 20 min at 4°C. 100µL cell-free supernatant containing extracellular protein (agarase) was poured into the prepared well of agar plate (1.5%) and stained with Lugol's iodine solution ( Scanning electron microscopy (SEM). The morphology and Cd uptake of the E.coli and P.aeruginosa BC15 were examined by SEM operating at an acceleration voltage of 10 kV equipped with Energy Dispersive X-ray analysis (EDAX). In order to prepare samples, a drop of E.coli and P.aeruginosa BC15 suspension (both untreated and treated with 1 mM CdCl 2 ) was mounted on aluminum stub and air dried. Bacterial cells were xed with 2.5% glutaraldehyde in phosphate buffer saline (pH 7.2), incubated at room temperature for 30 min and washed thrice with Na-phosphate buffer. Samples were dehydrated with graded acetone series (30, 50, 70, 80, 90 and 100%) for 10 min for each step. For better contrast, samples were coated with gold lm by sputter coater (Denton, Desk V HP) operating at 40 mA for 30 s under vacuum and analyzed by SEM (SEM-JEOL JSM-6400) equipped with Oxford energy dispersive Xray microanalysis system. CBOWCB immobilization on agar gel. The modi ed Wang et al (2014) method was used for Pseudomonas immobilization in agar. A 0.1 to 0.5% of agar solutions were prepared in PBS (pH 7.2) by warming them at 50°C. After cooling down to room temperature, a 2 to 20mL of cells was mixed with 190 mL different concentrations of agar solution and immediately 200mL was poured on 96 well plates.
After solidi cation at room temperature, the plates were stored in PBS (pH 7.2) and at 4°C. Immobilized cells agarase activity was measured in same with above mentioned section (Quantitative detection of Cd by CBOWCB).
Application of the CBOWCB. Water samples were collected from different polluted area in the southern part of Tamilnadu (Five sites from in and around Madurai, Tamilnadu). The water samples were ltered using Whatman lter paper and sterilized by autoclave. 10mL of each sample was added with the cultures (CBOWCB) in TY broth and incubated at 37°C in an orbital shaker at 225 rpm. The culture broth was harvested by centrifugation at 14,000 rpm for 20 min at 4 o C. The Cd content in the samples and CdCl 2 standards (0.1 ppm to 500ppm) were quanti ed as described in sections (Qualitative detection of Cd by CBOWCB and Quantitative detection of Cd by CBOWCB) and the results were analyzed by interception in the standard curve. The results were compared with the ICP-MS data, and the processing procedure was standardized for higher e ciency.
After solidi cation at room temperature, the plates were stored in PBS (pH 7.2) and at 4°C. Immobilized cells agarase activity was measured in same with above mentioned section (Quantitative detection of Cd by CBOWCB).
Application of the CBOWCB. Water samples were collected from different polluted area in the southern part of Tamilnadu (Five sites from in and around Madurai, Tamilnadu). The water samples were ltered using Whatman lter paper and sterilized by autoclave. 10mL of each sample was added with the cultures (CBOWCB) in TY broth and incubated at 37°C in an orbital shaker at 225 rpm. The culture broth was harvested by centrifugation at 14,000 rpm for 20 min at 4 o C. The Cd content in the samples and CdCl 2 standards (0.1 ppm to 500ppm) were quanti ed as described in sections (Qualitative detection of Cd by CBOWCB and Quantitative detection of Cd by CBOWCB) and the results were analyzed by interception in the standard curve. The results were compared with the ICP-MS data, and the processing procedure was standardized for higher e ciency.
BioIPN0, agarose, and latex,
together with a PP fibrous membrane control, were subjected to biodegradability
testing. A dual-enzyme system was needed to degrade the IPN consisting
of agarase (to degrade the agarose)26 (link) and
of Laccase (to degrade the latex).27 (link) Samples
of 1 cm2 size were treated in 10 mL of agarase at a concentration
of 0.625 U mL–1 in 50 mM Tris–HCl buffer
of pH 8.0 for 7 days. The biodegradation was carried out inside an
IKA KS 4000 incubator shaker at 40 °C and 60 rpm. After 1 week,
the remaining samples were washed with deionized water, dried, and
subjected to the second enzyme treatment. The samples were placed
in 10 mL of 1 U mL–1 Laccase in 10 mM acetate buffer
at pH of 5. The Laccase treatment was performed at 37 °C at 60
rpm using the same incubator shaker for 7 days. Following the biodegradability
test, the remaining membranes were collected on an NL 17 polyamide
membrane filter having a pore size of 0.45 μm (Whatman, GE Healthcare
Life Sciences) and thoroughly washed with deionized water. Then, the
membranes were dried in an oven at 40 °C under a vacuum for a
week. The weights of the dry samples before the start of the treatments
and at the end of the second enzyme test were recorded, and the weight
loss was calculated based on these values. The biodegradation experiments
were carried out in duplicate using independently prepared BioIPN0 membranes.
together with a PP fibrous membrane control, were subjected to biodegradability
testing. A dual-enzyme system was needed to degrade the IPN consisting
of agarase (to degrade the agarose)26 (link) and
of Laccase (to degrade the latex).27 (link) Samples
of 1 cm2 size were treated in 10 mL of agarase at a concentration
of 0.625 U mL–1 in 50 mM Tris–HCl buffer
of pH 8.0 for 7 days. The biodegradation was carried out inside an
IKA KS 4000 incubator shaker at 40 °C and 60 rpm. After 1 week,
the remaining samples were washed with deionized water, dried, and
subjected to the second enzyme treatment. The samples were placed
in 10 mL of 1 U mL–1 Laccase in 10 mM acetate buffer
at pH of 5. The Laccase treatment was performed at 37 °C at 60
rpm using the same incubator shaker for 7 days. Following the biodegradability
test, the remaining membranes were collected on an NL 17 polyamide
membrane filter having a pore size of 0.45 μm (Whatman, GE Healthcare
Life Sciences) and thoroughly washed with deionized water. Then, the
membranes were dried in an oven at 40 °C under a vacuum for a
week. The weights of the dry samples before the start of the treatments
and at the end of the second enzyme test were recorded, and the weight
loss was calculated based on these values. The biodegradation experiments
were carried out in duplicate using independently prepared BioIPN0 membranes.
In a previous study, a GFP-based biosensor utilizing the PcadR promoter was constructed (Rajkumar et al. 2018b ). The promoter-less plasmid, pUCP18 was generously provided by Prof. Pradeep Singh at the University of Washington. A 213-bp cassette containing the sensor element (P cadR promoter) (Accession number KU302252) for Cd was ampli ed from P. aeruginosa BC15 using oligonucleotides P cadR F1/P cadR R1 and P cadR F2/P cadR R2 (supplementary Table S2) as primers. The ampli ed Cd sensing promoter fragment was then digested with the restriction endonucleases PciI and KpnI, and inserted at the corresponding restriction sites into the promoterless pUCP18 plasmid. The Cd sensing promoter fragment (P cadR ) from the constructed plasmid (carrying pUCP-P cadR -gfp mut2 ) was con rmed by restriction digestion and BLAST analysis in the NCBI database.
The reporter gene β-agarase (dagA) was ampli ed from the vector pET21a, provided by Prof. In-Geol Choi at Korea University, Korea, using dagAF2 and dagAR2 primers containing the KpnI and HindIII sites, respectively. The 938 bp dagA gene PCR product was digested with KpnI and HindIII and ligated into the KpnI and HindIII digested pUCP-P cadR vector (gfp mut2 was digested using KpnI and HindIII). The nal constructs, pUCP-P cadR -dagA39 (GSR39) and pUCP-P cadR -dagA40 (GSR40), which carry P cadR (213bp) and the reporter gene agarase (dagA) (938 bp), were con rmed by colony PCR, restriction analysis, and sequencing using primers P cadR F2 and dagAR2 (Supplementary Table S2).
To enhance Cd resistance and reduce cell death, the expression vectors were electroporated into the expression host P. aeruginosa BC15.
The reporter gene β-agarase (dagA) was ampli ed from the vector pET21a, provided by Prof. In-Geol Choi at Korea University, Korea, using dagAF2 and dagAR2 primers containing the KpnI and HindIII sites, respectively. The 938 bp dagA gene PCR product was digested with KpnI and HindIII and ligated into the KpnI and HindIII digested pUCP-P cadR vector (gfp mut2 was digested using KpnI and HindIII). The nal constructs, pUCP-P cadR -dagA39 (GSR39) and pUCP-P cadR -dagA40 (GSR40), which carry P cadR (213bp) and the reporter gene agarase (dagA) (938 bp), were con rmed by colony PCR, restriction analysis, and sequencing using primers P cadR F2 and dagAR2 (Supplementary Table S2).
To enhance Cd resistance and reduce cell death, the expression vectors were electroporated into the expression host P. aeruginosa BC15.
Top products related to «Agarase»
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β-agarase is an enzyme that hydrolyzes the β-1,4-glycosidic bonds in agarose, a polysaccharide derived from certain red algae. It is used to break down agarose for various applications, such as the extraction of nucleic acids from agarose gels.
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Agarase is a laboratory enzyme used to digest and break down agarose, a polysaccharide derived from red algae. Agarase catalyzes the hydrolysis of agarose, resulting in the release of smaller oligosaccharides. This enzyme is commonly used in various molecular biology and biotechnology applications that involve the manipulation and analysis of DNA and other biomolecules.
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β-Agarase I is an enzyme that cleaves the glycosidic bonds in agarose, a polysaccharide derived from red algae. It is used to break down agarose gels, which are commonly used in various biotechnology applications, such as gel electrophoresis and affinity chromatography.
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Rat anti-BrdU antibody is a laboratory reagent used to detect the incorporation of the synthetic nucleoside bromodeoxyuridine (BrdU) into the DNA of proliferating cells. It can be used for applications such as cell proliferation assays and DNA synthesis studies.
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Phusion High-Fidelity DNA Polymerase is a thermostable DNA polymerase engineered for high-fidelity DNA amplification. It possesses 3'→5' exonuclease proofreading activity, resulting in an error rate up to 50-fold lower than Taq DNA polymerase.
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Silica gel 60 TLC plates are a type of thin-layer chromatography (TLC) plates used for separation and analysis of chemical compounds. They consist of a thin layer of silica gel, a porous form of silicon dioxide, coated on a solid support, typically a plastic or aluminum sheet. The silica gel 60 designation refers to the specific particle size and pore characteristics of the silica gel used in the plates. These TLC plates provide a stable and inert stationary phase for the separation of complex mixtures through differences in the relative affinities of the sample components for the stationary phase and the mobile phase.
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Agarase is a laboratory enzyme that hydrolyzes agarose, a polysaccharide extracted from red algae. It is commonly used in molecular biology and biochemistry applications for the separation and purification of nucleic acids and other macromolecules.
Sourced in United Kingdom, United States, China
Ab6326 is a primary antibody. It is a rabbit polyclonal antibody that recognizes the target antigen. The antibody is purified and suitable for use in various immunoassays.
Sourced in United States, Germany, United Kingdom, France, Spain, China, Sao Tome and Principe, Switzerland, Macao, Australia, Canada, Belgium, Japan, Israel, Poland
BrdU is a synthetic nucleoside that is an analog of the DNA base thymidine. It can be incorporated into the newly synthesized DNA of replicating cells, substituting for thymidine during the DNA synthesis phase of the cell cycle.
Sourced in United States
GTG low melting temperature agarose is a laboratory product used for in-gel cloning applications. It is designed to have a low melting point, enabling efficient DNA extraction and manipulation within the gel matrix.
More about "Agarase"
Agarase is an essential enzyme that plays a crucial role in the degradation of agar, a gelatin-like substance widely used in microbiology and biotechnology.
This hydrolytic enzyme catalyzes the breakdown of agarose, a complex polysaccharide derived from certain red algae.
Agarase is produced by various marine bacteria and fungi, and it has numerous applications, including the extraction and purification of nucleic acids, as well as the development of novel biomaterials.
Closely related to agarase is the β-agarase enzyme, which also catalyzes the hydrolysis of agarose.
Researchers studying agarase often employ techniques such as the use of Rat anti-BrdU antibody and Phusion High-Fidelity DNA Polymerase to investigate its structure, function, and potential applications.
The PubCompare.ai tool can enhance the reproducibility of agarase research by helping scientists quickly identify the best experimental protocols from the literature, preprints, and patents using advanced AI analysis.
This powerful tool can assist in locating optimal products and procedures, such as Silica gel 60 TLC plates and Ab6326 antibody, to streamline agarase experiments and obtain more reliable, reproducible results.
Understanding the role of agarase is crucial for diverse fields, including food processing, pharmaceuticals, and environmental remediation.
The insights gained from studying this enzyme can contribute to the development of novel biomaterials and innovative applications in biotechnology and beyond.
This hydrolytic enzyme catalyzes the breakdown of agarose, a complex polysaccharide derived from certain red algae.
Agarase is produced by various marine bacteria and fungi, and it has numerous applications, including the extraction and purification of nucleic acids, as well as the development of novel biomaterials.
Closely related to agarase is the β-agarase enzyme, which also catalyzes the hydrolysis of agarose.
Researchers studying agarase often employ techniques such as the use of Rat anti-BrdU antibody and Phusion High-Fidelity DNA Polymerase to investigate its structure, function, and potential applications.
The PubCompare.ai tool can enhance the reproducibility of agarase research by helping scientists quickly identify the best experimental protocols from the literature, preprints, and patents using advanced AI analysis.
This powerful tool can assist in locating optimal products and procedures, such as Silica gel 60 TLC plates and Ab6326 antibody, to streamline agarase experiments and obtain more reliable, reproducible results.
Understanding the role of agarase is crucial for diverse fields, including food processing, pharmaceuticals, and environmental remediation.
The insights gained from studying this enzyme can contribute to the development of novel biomaterials and innovative applications in biotechnology and beyond.