The strains, plasmids, and primers used in this study are listed in Table S2. The E. coli TOP10 strain (Thermo Fisher Scientific, USA) was used for gene cloning. E. coli was routinely cultured in Luria-Bertani (LB) medium supplemented with appropriate antibiotics (10 μg/mL streptomycin or 50 μg/mL ampicillin) at 37 °C in a shaking incubator (Jeiotech, Korea). The genes for MBP, GST, NEXT, GFP and taCA were cloned by polymerase chain reaction (PCR) using pMAL-c5X (New England Biolabs, USA), pGEX-4T-1 (GE Healthcare, USA), pET-hmCA (9), pTH-GFP (36) , and pET-taCA (19) as the templates. The primers for the solubility tags contain the sequence for flexible linker (GGGGS) 2 along with NdeI and NcoI restriction sites. The PCR fragments were cloned into the pGEM-T Easy vector (Promega, USA) and the amplified sequences were confirmed by direct sequencing. The genes for the Fh8 tag (GenBank accession number: AF213970), hEGF (GenBank accession number: M15672) and isPETase (GenBank accession number: 6EQD_A) were chemically synthesized along with the linker sequence (only for Fh8) and the restriction sites (Genotech, Korea). The genes were subcloned into pET-22b(+) (Novagen, USA). All of the recombinant genes have a hexahistidine (His 6 )-tag sequence at their 3' termini provided by the parent vector.
Shaking incubator
Manufactured by Jeio Tech
Sourced in Cameroon
The Shaking Incubator is a laboratory equipment designed to simultaneously incubate and agitate samples. It maintains a controlled temperature environment while consistently mixing the contents to ensure uniform conditions for cell culture, bacterial growth, or other applications requiring temperature and mixing.
Automatically generated - may contain errors
Lab products found in correlation
Pgex 4t 1, by GE Healthcare (1 mentions)
Pmal c5x, by New England Biolabs (1 mentions)
Top 10 e coli strain, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Electroporator, by Eppendorf (1 mentions)
Gel scanner, by Epson (1 mentions)
Benchtop cooling centrifuge, by Eppendorf (1 mentions)
Low molecular weight markers, by Cytiva (1 mentions)
Ultrospec 2100 pro spectrophotometer, by Cytiva (1 mentions)
Superdex 75, by Cytiva (1 mentions)
Akta purification system, by Cytiva (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Bio Basic (1 mentions)
Benzonase, by Merck Group (1 mentions)
Chicken egg lysozyme, by Thermo Fisher Scientific (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Ampicillin, by Bio Basic (1 mentions)
6 protocols using shaking incubator
1
Cloning and Expression of Recombinant Proteins
2
Disintegration Time Study of DNA Capsules
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The disintegration time study was based on Ph Pol XI 1 with some modifications due to the physicochemical properties of the active substance (DNA). According to the definition in Ph Pol XI, a complete disintegration is defined as the state where all remainders of the capsule are a soft mass without a compact, non-wetted core, except for fragments of undissolved capsule shell. According to the indications of the Ph Pol XI, 1 6 capsules were tested at one time. The temperature of the liquid was maintained at 37°C ±0.2°C (water or 0.1 M hydrochloric acid solution) and the test time did not exceed 30 min. The capsules were placed in 250 mL beakers containing 100 mL of water or hydrochloric acid solution (pH 1.5) in a shaking incubator (Jeio Tech, Daejeon, South Korea) at 37°C and 100 rpm. The capsules were observed and the appearance of the capsules and the time needed for disintegration were assessed (Fig. 2). Additionally, the disintegration time experiment was carried out at a temperature 2°C lower than that recommended by the Ph Pol XI. This variant of the study at 35°C allowed for more accurate tracking of capsule disintegration and the preparation of appropriate photographic documentation (Fig. 2).
3
In Vitro Evaluation of Gene Capsule Dissolution
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Test conditions similar to the nature and specificity of the active substance, i.e., plasmid DNA, were created. Water was used as the dissolution medium for the polymerase chain reaction (PCR) method of DNA detection. The level of HCl, phosphate buffer or NaOH recommended in the Ph Pol XI would disrupt the PCR process. Capsules were prepared as described in the "Formulation and preparation of gene capsules" section. Single capsules were placed in 250 mL beakers containing 100 mL water and placed in a shaking incubator (Jeio Tech) at 37°C and 100 rpm. Samples (8 µL) were collected every 30 s for 10 min and after 30 min (Fig. 3). A total of 20 capsules were tested (numbered from 1-20); the 15 th capsule exceeded the allowable deviation and the 16 th capsule was at the limit of the allowable deviation. X -the average weight of lactose; deviation [%] -deviation expressed as a percentage of the difference from the average mass of capsules.
Values in bold slightly exceeded or were at the limit of the allowable deviation, according to Polish Pharmacopoeia XI (Ph Pol XI) 1 .
Values in bold slightly exceeded or were at the limit of the allowable deviation, according to Polish Pharmacopoeia XI (Ph Pol XI) 1 .
4
Expression and Purification of cHSPA6
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The ORF of cHSPA6 cloned on pET15 vector and expression host E. coli BL21 (DE3) pLysS were kindly provided by Elrobh et al. (2011) (link). IPTG and ampicillin were obtained from Biobasic. Benzonase was purchased from Sigma, Chicken egg lysozyme from USB Corporation. Superdex 75, Ni–NTA resin, low molecular weight markers and prepacked columns were from Amersham Biosciences. All other chemicals used in this study were of reagent grade. Ultrospec 2100 pro Spectrophotometer, AKTA purification system, SDS–PAGE assembly were from Amersham Biosciences. Thermomixer, electroporator and benchtop cooling centrifuge were from Eppendorf. Lamp sterilizer from Cole-Parmer, shaking incubator from Jeio Tech, South Korea, gel scanner from Epson and pH meter was from Sentron.
5
Enzymatic Hydrolysis of Milled Biomass
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The buffer solutions used in planetary milling were suitable for cellulase activity. Hence, enzymatic hydrolysis process was continually carried out after the planetary milling without washing or exchanging buffer. A cellulase cocktail (Worthington Biochemical Co., USA) was added into the buffer containing milled biomass. Enzymatic hydrolysis was performed at 50°C in a shaking incubator (JEIO TECH, Rep Korea) at 200 rpm. The glucose yield was calculated using the following equation: which was used in a previous study [7 (link)].
6
Enzymatic Esterification of Formic Acid
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Theoretically, the esterification of 1 mol of formic acid with 1 mol of phenethyl alcohol produces 1 mol of phenethyl formate and 1 mol of water. It is therefore a 1:1 stoichiometric reaction. As such, formic acid (100 mM) and phenethyl alcohol (100 mM) were dissolved in a given solvent, and 10 mL of each solution was transferred to a 50 mL serum bottle. The total reaction volume was 20 mL with a final substrate concentration of 50 mM. Prior to the reaction, an immobilized lipase was added to the solution. As the volatilities of the solvent, the substrates, and the products can influence the reaction, the serum bottle was sealed by crimping. The reaction was then mixed for 4 h at 150 rpm using a shaking incubator (JEIO TECH.CO., LTD, Daejeon, Korea).
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