Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli
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{"title"=>"Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli", "type"=>"journal", "authors"=>[{"first_name"=>"Sushila", "last_name"=>"Maharjan", "scopus_author_id"=>"24069223400"}, {"first_name"=>"Bijay", "last_name"=>"Singh", "scopus_author_id"=>"57188671129"}, {"first_name"=>"Jin Duck", "last_name"=>"Bok", "scopus_author_id"=>"7006983504"}, {"first_name"=>"Jeong In", "last_name"=>"Kim", "scopus_author_id"=>"56181604300"}, {"first_name"=>"Tao", "last_name"=>"Jiang", "scopus_author_id"=>"54683986000"}, {"first_name"=>"Chong Su", "last_name"=>"Cho", "scopus_author_id"=>"55430062200"}, {"first_name"=>"Sang Kee", "last_name"=>"Kang", "scopus_author_id"=>"8313343600"}, {"first_name"=>"Yun Jaie", "last_name"=>"Choi", "scopus_author_id"=>"35953402000"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"24809485", "sgr"=>"84901507331", "doi"=>"10.1371/journal.pone.0096259", "scopus"=>"2-s2.0-84901507331", "pui"=>"373185885", "issn"=>"19326203"}, "id"=>"3335e07d-c342-351c-bd70-3b859a78863b", "abstract"=>"Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodology to optimize the soluble expression of mRANKL in E. coli. We optimized the codon usage of mRANKL sequence to a preferred set of codons for E. coli changing its codon adaptation index from 0.64 to 0.76, tending to increase its expression level in E. coli. Further, we utilized central composite design to predict the optimum combination of variables (cell density before induction, lactose concentration, post-induction temperature and post-induction time) for the expression of mRANKL. Finally, we investigated the effects of various experimental parameters using response surface methodology. The best combination of response variables was 0.6 OD600, 7.5 mM lactose, 26°C post-induction temperature and 5 h post-induction time that produced 52.4 mg/L of fusion mRANKL. Prior to functional analysis of the protein, we purified mRANKL to homogeneity and confirmed the existence of trimeric form of mRANKL by native gel electrophoresis and gel filtration chromatography. Further, the biological activity of mRANKL to induce osteoclast formation on RAW264.7 cells was confirmed by tartrate resistant acid phosphatase assay and quantitative real-time polymerase chain reaction assays. Importantly, a new finding from this study was that the biological activity of mRANKL is higher than its extracellular counterpart. To the best of our knowledge, this is the first time to report heterologous expression of mRANKL in soluble form and to perform a comparative study of functional properties of both forms of RANKL.", "link"=>"http://www.mendeley.com/research/exploring-codon-optimization-response-surface-methodology-express-biologically-active-transmembrane", "reader_count"=>15, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>5, "Student > Ph. D. Student"=>3, "Student > Master"=>2, "Student > Bachelor"=>1, "Professor"=>1, "Other"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>5, "Student > Ph. D. Student"=>3, "Student > Master"=>2, "Student > Bachelor"=>1, "Professor"=>1, "Other"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>8, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Chemical Engineering"=>1, "Chemistry"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Chemistry"=>{"Chemistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>8}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"Netherlands"=>1, "Korea (South)"=>1, "Japan"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1489724"], "description"=>"<p>Three dimensional surface plots of combined effect of cell density (OD<sub>600</sub>) and lactose concentration (A), OD<sub>600</sub> and post-induction temperature (B), OD<sub>600</sub> and post-induction time (C), post-induction temperature and lactose concentration (D), post-induction time and lactose concentration (E) and post- induction temperature and post-induction time (F) on mRANKL production.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "visualize"], "article_id"=>1020320, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g002", "stats"=>{"downloads"=>4, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Response_surface_plots_to_visualize_the_relationship_between_the_response_and_experimental_levels_of_each_factor_/1020320", "title"=>"Response surface plots to visualize the relationship between the response and experimental levels of each factor.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489726"], "description"=>"<p>A: Comassie blue stained SDS gel showing lysates from two SHuffle <i>E. coli</i>-pOmR-c5X colonies expressing the mRANKL fusion proteins (∼75 kDa). Lane 1: Protein standards, molecular masses are indicated in kilodaltons (kDa); Lanes 2 and 3: Insoluble and soluble fractions from clone 1, respectively; Lanes 4 and 5: Insoluble and soluble fractions from clone 2, respectively. B: Comassie blue stained SDS gel showing purification of mRANKL using amylose resin. Lane 1: crude fusion mRANKL; Lane 2: Flow through; Lane 3: Protein standards, molecular masses are indicated in kilodaltons (kDa); Lanes 4–6: Elution fractions. C: Western blot analysis of mRANKL. Different amounts of purified fusion mRANKL was run in 4–20% SDS gel and transferred onto a nitrocellulose membrane. Detection was performed with anti-RANKL primary antibody, goat IgG HRP-conjugated secondary antibody and chemiluminescent substrate. Lane 1: Protein marker; Lanes 2–5: 30, 50, 100, and 200 ng of purified fusion mRANKL protein respectively.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "blot", "mrankl", "fusion"], "article_id"=>1020322, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g003", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SDS_PAGE_and_Western_blot_analyses_of_mRANKL_fusion_proteins_/1020322", "title"=>"SDS-PAGE and Western blot analyses of mRANKL fusion proteins.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489733"], "description"=>"<p>RAW 246.7 cells were treated with each protein at the concentration of 100/ml for 6 days and TRAP-positive osteoclasts were visualized under microscope. mRANKL (A); RANKL-Ex (B); Untreated RAW 246.7 cells (C).</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "staining"], "article_id"=>1020328, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g005", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TRAP_staining_assay_/1020328", "title"=>"TRAP staining assay.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489728"], "description"=>"<p>A.SDS-PAGE analysis of purified mRANKL. The fusion mRANKL was cleaved by Factor Xa, purified by affinity chromatography followed by gel filtration chromatography. Eluted proteins fractions (Lane 2–7) were checked by SDS-PAGE gel. Lane 1: Protein standards, molecular masses are indicated in kilodaltons (kDa). B. Native PAGE analysis. PAGE analysis demonstrated the native state of mRANKL (molecular weight of approximately 100 kDa), consistent with the size of a homotrimer. Lane 1: Protein standard; Lane 2: mRANKL. C. Gel filtration chromatography profile. The elution peaks at 14.90 ml and 20.09 ml correspond to estimated molecular masses of ∼97 and ∼44 kDa, based on the elution volumes of known molecular standards. The estimated molecular masses are close to the predicted molecular weight of trimeric form (∼104 kDa) and monomeric form of the protein (∼35 kDa). The y axis indicates the absorption at 280 nm in milliabsorbance units (mAU) and the x axis indicates the volume in milliliters (ml) passed over the column.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "mrankl", "affinity", "chromatography", "gel", "filtration"], "article_id"=>1020324, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g004", "stats"=>{"downloads"=>0, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Purification_of_mRANKL_by_affinity_chromatography_and_gel_filtration_chromatography_/1020324", "title"=>"Purification of mRANKL by affinity chromatography and gel filtration chromatography.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489810", "https://ndownloader.figshare.com/files/1489811", "https://ndownloader.figshare.com/files/1489812", "https://ndownloader.figshare.com/files/1489813", "https://ndownloader.figshare.com/files/1489814", "https://ndownloader.figshare.com/files/1489815", "https://ndownloader.figshare.com/files/1489817"], "description"=>"<div><p>Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodology to optimize the soluble expression of mRANKL in <i>E. coli</i>. We optimized the codon usage of mRANKL sequence to a preferred set of codons for <i>E. coli</i> changing its codon adaptation index from 0.64 to 0.76, tending to increase its expression level in <i>E. coli</i>. Further, we utilized central composite design to predict the optimum combination of variables (cell density before induction, lactose concentration, post-induction temperature and post-induction time) for the expression of mRANKL. Finally, we investigated the effects of various experimental parameters using response surface methodology. The best combination of response variables was 0.6 OD<sub>600</sub>, 7.5 mM lactose, 26°C post-induction temperature and 5 h post-induction time that produced 52.4 mg/L of fusion mRANKL. Prior to functional analysis of the protein, we purified mRANKL to homogeneity and confirmed the existence of trimeric form of mRANKL by native gel electrophoresis and gel filtration chromatography. Further, the biological activity of mRANKL to induce osteoclast formation on RAW264.7 cells was confirmed by tartrate resistant acid phosphatase assay and quantitative real-time polymerase chain reaction assays. Importantly, a new finding from this study was that the biological activity of mRANKL is higher than its extracellular counterpart. To the best of our knowledge, this is the first time to report heterologous expression of mRANKL in soluble form and to perform a comparative study of functional properties of both forms of RANKL.</p></div>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "exploring", "codon", "optimization", "methodology", "biologically", "transmembrane", "rankl"], "article_id"=>1020392, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0096259.s001", "https://dx.doi.org/10.1371/journal.pone.0096259.s002", "https://dx.doi.org/10.1371/journal.pone.0096259.s003", "https://dx.doi.org/10.1371/journal.pone.0096259.s004", "https://dx.doi.org/10.1371/journal.pone.0096259.s005", "https://dx.doi.org/10.1371/journal.pone.0096259.s006", "https://dx.doi.org/10.1371/journal.pone.0096259.s007"], "stats"=>{"downloads"=>14, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Exploring_Codon_Optimization_and_Response_Surface_Methodology_to_Express_Biologically_Active_Transmembrane_RANKL_in_E_coli_/1020392", "title"=>"Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in <i>E. coli</i>", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489739"], "description"=>"<p>TRAP activity was determined in the medium of RAW264.7 cells treated with mRANKL (30–200 ng/ml) for 6 days, numerical value with mRANKL denotes the concentration of mRANKL used (A); RAW264.7 cells were treated with 100 ng/ml of each mRANKL and RANKL-Ex for 6 days (B).</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "osteoclastogenesis", "rankl"], "article_id"=>1020331, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g006", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Induction_of_osteoclastogenesis_by_RANKL_in_RAW264_7_cells_/1020331", "title"=>"Induction of osteoclastogenesis by RANKL in RAW264.7 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489742"], "description"=>"<p>The expression of osteoclast differentiation (A–C) and functional markers (D, E) were studied at day 6 after mRANKL (100 ng/ml) and RANKL-Ex (100 ng/ml) exposure to RAW 246.7 cells. The results of the mRNA levels of genes of interest were normalized to GAPDH (control gene) and plotted. The data shown are representatives of three different experiments.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "rt-pcr"], "article_id"=>1020334, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g007", "stats"=>{"downloads"=>0, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Quantitative_RT_PCR_analyses_/1020334", "title"=>"Quantitative RT-PCR analyses.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489743"], "description"=>"<p>Primers used in this study.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models"], "article_id"=>1020335, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.t001", "stats"=>{"downloads"=>3, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primers_used_in_this_study_/1020335", "title"=>"Primers used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489744"], "description"=>"<p>The independent variables and coded values of variables used in central composite rotary design.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "variables", "coded", "composite", "rotary"], "article_id"=>1020336, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.t003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_independent_variables_and_coded_values_of_variables_used_in_central_composite_rotary_design_/1020336", "title"=>"The independent variables and coded values of variables used in central composite rotary design.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489745"], "description"=>"<p>Bacterial strains and plasmids used in this study.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "strains", "plasmids"], "article_id"=>1020337, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.t002", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bacterial_strains_and_plasmids_used_in_this_study_/1020337", "title"=>"Bacterial strains and plasmids used in this study.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489746"], "description"=>"<p>A  =  OD<sub>600</sub>; B  =  Lactose concentration; C  =  Temperature; D  =  Induction time; df  =  Degrees of freedom.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "quadratic", "mrankl"], "article_id"=>1020338, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.t005", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ANOVA_for_response_surface_quadratic_model_for_mRANKL_production_/1020338", "title"=>"ANOVA for response surface quadratic model for mRANKL production.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489747"], "description"=>"<p>CCD with measured and predicted response of mRANKL production.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "mrankl"], "article_id"=>1020339, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.t004", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CCD_with_measured_and_predicted_response_of_mRANKL_production_/1020339", "title"=>"CCD with measured and predicted response of mRANKL production.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489748"], "description"=>"<p>Confirmation experiments for mRANKL production.</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "experiments", "mrankl"], "article_id"=>1020340, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.t006", "stats"=>{"downloads"=>3, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Confirmation_experiments_for_mRANKL_production_/1020340", "title"=>"Confirmation experiments for mRANKL production.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-05-08 03:07:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/1489722"], "description"=>"<p>Correlation between predicted and actual value for mRANKL production (A). The studentized and normal percentage probability plot of mRANKL production (B).</p>", "links"=>[], "tags"=>["Biochemistry", "dna", "DNA amplification", "DNA modification", "DNA recombination", "DNA synthesis", "proteins", "Recombinant proteins", "Nucleic acids", "biophysics", "biotechnology", "cell biology", "Molecular cell biology", "Computational biology", "genetics", "gene expression", "Protein translation", "Molecular genetics", "microbiology", "Medical microbiology", "Microbial pathogens", "Bacterial pathogens", "Escherichia coli", "molecular biology", "Molecular biology techniques", "Sequencing techniques", "Sequence analysis", "Synthetic Biology", "Model organisms", "Prokaryotic models", "estimating", "adequacy", "regression"], "article_id"=>1020318, "categories"=>["Biological Sciences"], "users"=>["Sushila Maharjan", "Bijay Singh", "Jin-Duck Bok", "Jeong-In Kim", "Tao Jiang", "Chong-Su Cho", "Sang-Kee Kang", "Yun-Jaie Choi"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0096259.g001", "stats"=>{"downloads"=>1, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Diagnostic_plots_for_estimating_the_adequacy_of_the_regression_model_/1020318", "title"=>"Diagnostic plots for estimating the adequacy of the regression model.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-05-08 03:07:56"}

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