Name: production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock
Uploaded: 2017-06-29T14:00:00
Description: Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

This work was supported by the National Natural Science Foundation of China (Grant No. 21576279, 20906076) and the KRIBB Research Initiative Program (KGM2211531).

1. Preparation of Bamboo Hydrolysate
<ol>
	<li>Add 5 g of bamboo powder to 40 mL of an NaOH solution to achieve a 10% (g/g) final concentration in a 250 mL flask. Use a series of NaOH solutions ranging from 0.05 M to 0.50 M in increments of 0.05 M.
	<ol>
		<li>Incubate the mixture for 60 min at 60 &#176;C or 100 &#176;C in a water bath. Incubate at 121 &#176;C in an autoclave.</li>
	</ol>
	</li>
	<li>After incubation, leave the mixture to stand for 4 h at room temperature. Then, remove the supe...

<ol>
	<li>Zeng, A. P., Biebl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+chemicals+from+biotechnology:+the+case+of+1,+3-propanediol+production+and+the+new+trends.">Bulk chemicals from biotechnology: the case of 1, 3-propanediol production and the new trends.</a> Adv Biochem Eng Biotechnol. 74, 239-259 (2002).</li><li>Wei, D., Wang, M., Jiang, B., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dihydroxyacetone+kinases+I+and+II+in+the+dha+regulon+of+Klebsiella+pneumoniae.">Role of dihydroxyacetone kinases I and II in the dha regulon of Klebsiella pneumoniae.</a> J Biotechnol. 177, 13-19 (2014).</li><li>Celi&#324;ska, E., Grajek, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotechnological+production+of+2,3-butanediol-current+state+and+prospects.">Biotechnological production of 2,3-butanediol-current state and prospects.</a> Biotechnol Adv. 27 (6), 715-725 (2009).</li><li>Chen, C., Wei, D., Shi, J., Wang, M., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+2,+3-butanediol+stereoisomer+formation+in+Klebsiella+pneumoniae.">Mechanism of 2, 3-butanediol stereoisomer formation in Klebsiella pneumoniae.</a> Appl Microbiol Biotechnol. 98 (10), 4603-4613 (2014).</li><li>Wei, D., Wang, M., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+recombinase+assisted+gene+replacement+in+Klebsiella+pneumoniae.">Red recombinase assisted gene replacement in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 39 (8), 1219-1226 (2012).</li><li>Wei, D., Sun, J., Shi, J., Liu, P., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+strategy+to+improve+efficiency+for+gene+replacement+in+Klebsiella+pneumoniae.">New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 40 (5), 523-527 (2013).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+RecBCD+in+Klebsiella+pneumoniae+by+Gam+and+its+effect+on+the+efficiency+of+gene+replacement.">Inhibition of RecBCD in Klebsiella pneumoniae by Gam and its effect on the efficiency of gene replacement.</a> J Basic Microbiol. 56 (2), 120-126 (2016).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-acetoin+accumulation+and+dissimilation+in+Klebsiella+pneumoniae.">R-acetoin accumulation and dissimilation in Klebsiella pneumoniae.</a> J Ind Microbiol Biotechnol. 42 (8), 1105-1115 (2015).</li><li>Wei, D., Xu, J., Sun, J., Shi, J., Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Ketogluconic+acid+production+by+Klebsiella+pneumoniae+CGMCC+1.6366.">2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366.</a> J Ind Microbiol Biotechnol. 40 (6), 561-570 (2013).</li><li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-stage+fermentation+for+2-ketogluconic+acid+production+by+Klebsiella+pneumoniae.">Two-stage fermentation for 2-ketogluconic acid production by Klebsiella pneumoniae.</a> J Microbiol Biotechnol. 24 (6), 781-787 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gluconic+acid+production+by+gad+mutant+of+Klebsiella+pneumoniae.">Gluconic acid production by gad mutant of Klebsiella pneumoniae.</a> World J Microbiol Biotechnol. 32 (8), 1-11 (2016).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+xylonic+acid+by+Klebsiella+pneumoniae.">Production of xylonic acid by Klebsiella pneumoniae.</a> Appl Microbiol Biotechnol. 100 (23), 10055-10063 (2016).</li><li>Kumar, P., Barrett, D. M., Delwiche, M. J., Stroeve, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+pretreatment+of+lignocellulosic+biomass+for+efficient+hydrolysis+and+biofuel+production.">Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production.</a> Ind Eng Chem Res. 48 (8), 3713-3729 (2009).</li><li>Brisse, S., Grimont, F., Grimont, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genus+Klebsiella.">The genus Klebsiella.</a> The Prokaryotes. , 159-196 (2006).</li><li>Hao, J., Lin, R., Zheng, Z., Liu, H., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+microorganisms+able+to+produce+1,+3-propanediol+under+aerobic+conditions.">Isolation and characterization of microorganisms able to produce 1, 3-propanediol under aerobic conditions.</a> World J Microbiol Biotechnol. 24 (9), 1731-1740 (2008).</li><li>Hong, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+alkaline+pretreatment+on+corn+stover+for+enhanced+production+of+1.3-propanediol+and+2,+3-butanediol+by+Klebsiella+pneumoniae+AJ4.">Optimization of alkaline pretreatment on corn stover for enhanced production of 1.3-propanediol and 2, 3-butanediol by Klebsiella pneumoniae AJ4.</a> Biomass Bioenerg. 77, 177-185 (2015).</li><li>Pienkos, P. T., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pretreatment+and+conditioning+processes+on+toxicity+of+lignocellulosic+biomass+hydrolysates.">Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates.</a> Cellulose. 16 (4), 743-762 (2009).</li></ol>

K. pneumoniae belongs to the genus Klebsiella in the family Enterobacteriaceae. K. pneumoniae is distributed widely in natural environments such as soil, vegetation, and water14. The wildtype K. pneumoniae strain used in this research was isolated from soil and is used for 1,3-propanediol production15. K. pneumoniae and mutants of this species produce many chemicals under different conditions.
<p class="jove_content"...

Klebsiella pneumoniae is a bacterium that grows well under both aerobic and anaerobic conditions. K. pneumoniae is an important industrial microorganism used to produce many chemicals. 1,3-propanediol is a valuable chemical that is mainly used as a monomer to synthesize polytrimethylene terephthalate. Polytrimethylene terephthalate is a biodegradable polyester that exhibits better properties than those of 1,2-propanediol, butanediol, or ethylene glycol1. 1,3-propanediol is produced by K. pneumoniae using glycerol as a substrate in oxygen-limited conditions2. 2,3-butanediol and its derivatives have applications in the field of plastics, solvent production, and synthetic rubber and have the potential to be used as biofuel3. With glucose as the substrate, 2,3-butanediol is the main metabolite of the wildtype strain4. 2,3-butanediol is synthesized from pyruvate. First, two molecules of pyruvate condense to yield &#945;-acetolactate; this reaction is catalyzed by &#945;-acetolactate synthase. &#945;-acetolactate is then converted to acetoin by &#945;-acetolactate decarboxylase. R-acetoin can be further reduced to 2,3-butanediol when catalyzed by butanediol dehydrogenase. An efficient gene replacement method suitable for K. pneumoniae has been explored, and many mutants have been constructed5,6,7. A budC mutant, which lost its 2,3-butanediol dehydrogenase activity, accumulates high levels of acetoin in culture broth. Acetoin is used as an additive to enhance the flavor of food8. When budA, which encodes &#945;-acetolactate decarboxylase, is mutated, 2-ketogluconic acid accumulates in the broth. 2-ketogluconic acid is used for the synthesis of erythorbic acid (isoascorbic acid), an antioxidant used in the food industry9. 2-ketogluconic acid is an intermediate of the glucose oxidation pathway; in this pathway, located in the periplasmic space, glucose is oxidized to gluconic acid and then further oxidized to 2-ketogluconic acid. Gluconic acid and 2-ketogluconic acid produced in the periplasm can be transported to the cytoplasm for further metabolism. The accumulation of 2-ketogluconic acid is dependent upon acidic conditions, and higher air supplementation favors 2-ketogluconic acid production10. Gluconate dehydrogenase, encoded by gad, catalyzes the conversion of gluconic acid to 2-ketolguconic acid. The gad mutant of K. pneumoniae produced high levels of gluconic acid instead of 2-ketogluconic acid, and this process is also dependent upon acidic conditions. Gluconic acid is a bulk organic acid and is used as an additive to increase the properties of cement11. Glucose oxidation to gluconic acid is catalyzed by glucose dehydrogenase. Xylose is also a suitable substrate of glucose dehydrogenase. When xylose is used as a substrate, K. pneumoniae produces xylonic acid12.
Chemical production using biomass as a feedstock is a hot topic in biotechnology13. The main components of biomass are cellulose, hemicellulose, and lignin. However, these macromolecular compounds cannot be directly catabolized by most microorganisms (including K. pneumoniae). Cellulose and hemicelluloses in the biomass must be hydrolyzed to glucose and xylose and can then be used by microorganisms. The presence of lignin in lignocelluloses creates a protective barrier that prevents biomass hydrolyzation by enzymes. Thus, a pretreatment process that removes lignin and hemicelluloses and reduces the crystallinity of cellulose is always performed during biomass utilization by microorganisms. Many pretreatment methods have been developed: acid, alkaline, ammonia, and steam pretreatments are common.
Bamboo is abundant in tropical and subtropical regions and is an important biomass resource. Here, the preparation of bamboo hydrolysate and chemical production using bamboo hydrolysate are presented

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">autoclave</td>
			<td style="width:136px;">SANYO</td>
			<td align="right" style="width:119px;">3780</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">bioreactor</td>
			<td style="width:136px;">Sartorius stedim biotech</td>
			<td style="width:119px;">Bostat Aplus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">agitator</td>
			<td style="width:136px;">IKA</td>
			<td style="width:119px;">RW 20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath shaker</td>
			<td style="width:136px;">Zhicheng</td>
			<td style="width:119px;">ZWY-110X50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">high performance liquid chromatograph system</td>
			<td style="width:136px;">Shimadzu Corp</td>
			<td style="width:119px;">20AVP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">centrifuge</td>
			<td style="width:136px;">Hitachi</td>
			<td style="width:119px;">CR22G III</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bamboo powder</td>
			<td style="width:136px;">purchased from Zhejiang Province, China</td>
			<td style="width:119px;"></td>
			<td>mesh number, 50</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">cellulase</td>
			<td style="width:136px;">Youtell Biochemical, Shandong, China</td>
			<td style="width:119px;"></td>
			<td>200 PFU/ml</td>
		</tr>
	</tbody>
</table>

production of chemicals by klebsiella pneumoniae using bamboo hydrolysate as feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

In this protocol, the bamboo was pretreated using alkali. The optimal incubation parameters-a temperature of 121 &#176;C and 0.25 M NaOH-were determined in Figures 1 and 2. The pretreated bamboo was enzymatically hydrolyzed, and the glucose and xylose concentrations obtained in the hydrolysate were measured. Higher temperatures favored sugar production, so 121 &#176;C was selected as the optimal temperature. The glucose and xylose produced in the hydrolys...

Watch this Scientific Journal Video about Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock at JoVE.com

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

The overall goal of this experiment is to observe production of the chemicals 2, 3-Butanediol, Acetoin, 2-ketogluconic acid and xylonic acid by Klebsiella pneumoniae using the hydrolysate of bamboo as the feedstock. To begin, using a series of sodium hydroxide solutions in 250 milliliter flasks, add five grams of bamboo powder to 40 milliliters of sodium hydroxide to make a 10%solution. Incubate the solutions in a water bath at 60 degrees Celsius, or 100 degrees Celsius for 60 minutes, or incubate the solutions in an autoclave at 121 degrees Celsius for 60 minutes.Following the incubation, centrifuge the mixtures for five minutes then remove the supernatant, add 100 milliliters of fresh water, and mix. Repeat the washing five to six times until the pH of the supernatant reaches 6.8, then to carry out enzymatic hydrolysis, use 98%sulfuric acid to adjust 50 milliliters of each solution to pH 5.0. Next add 0.5 milliliters of 200 filter paper activity, or FPU, per milliliter of cellulase.The final ratio of cellulase to bamboo should be 20 FPU per one gram of bamboo. Incubate the mixture at 50 degrees Celsius with shaking for 36 hours. Following hydrolysis, some solid will remain in the mixture.To obtain a clear hydrolysate, centrifuge the solutions at 7, 690 times g for five minutes. To prepare a large volume of hydrolysate, follow a similar procedure as just demonstrated, except replace the 250 milliliter flasks with a 30 liter stainless steel tank, a 60 liter plastic tank, and a modified shaking water bath which is equipped with a mechanical agitator. After preparing fermentation medium according to the text protocol, prepare a seed culture by adding K-pneumoniae cells to a plate, and culture overnight, then transfer the culture to a 250 milliliter flask containing 50 milliliters of LB medium.Incubate the culture at 37 degrees Celsius and 200 RPM overnight. Wild-type, the bud C mutant and the bud A mutant are used for 2, 3-Butanediol, Acetoin, and 2-ketogluconic acid production respectively. Inoculate 50 milliliters of the seed culture into the bioreactor.Maintain the culture at 37 degrees Celsius with a pH of 6.0 and 7.0 for Acetoin and 2, 3-Butanediol respectively. For 2-ketogluconic acid production, maintain the culture at pH 7.0 for the first four hours, then switch to a pH of 5.0. For 2, 3-Butanediol production, maintain the culture in a microaerobic environment with air supplementation at two liters per minute and 250 RPM.For Acetoin production, maintain the culture in an aerobic environment with air supplementation at four liters per minute at 450 RPM. For 2-ketogluconic acid production, maintain high aerobic conditions with air supplementation and agitation rates at four liters per minute, and 500 RPM respectively. Finally collect five milliliter samples every two hours during the fermentation, and use high-pressure liquid chromatography to analyze them to determine the chemical concentrations in the broth.Using the protocol demonstrated in this video, higher temperatures and sodium hydroxide concentrations between 0.25 and 0.40 molar favored production of hydrolysate from bamboo. About 20 grams per liter of glucose and 10 grams per liter of xylose were produced during the flask scale enzymatic hydrolysis and 30 grams per liter of glucose and 15 grams per liter of xylose were obtained from the large volume hydrolysate preparation. As shown here, 2, 3-Butanediol was produced by K-pneumoniae in microaerobic conditions.The process was divided into two periods, in the first glucose was used by the cells to produce 7.6 grams per liter of 2, 3-Butanediol. In the second period, the xylose in the broth was used by the cells, and an additional 5.1 grams per liter of 2, 3-Butanediol was produced. Acetoin was produced by the bud C mutant of K-pneumoniae under aerobic conditions.As in 2, 3-Butanediol production by the wild-type strain, glucose and then xylose were used in sequence and K-pneumoniae delta bud C produced 13 grams per liter of Acetoin. 2-ketogluconic acid and xylonic acid were produced by the bud A mutant of K-pneumoniae, and this process required high air supplementation. The glucose in the medium was first converted to gluconic acid, and further converted to 2-ketogluconic acid.At the same time, xylose was converted to xylonic acid. 25 grams per liter of 2-ketogluconic acid and 14 grams per liter of xylonic acid were finally produced.

production-chemicals-klebsiella-pneumoniae-using-bamboo-hydrolysate

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The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...