This document describes a new method for efficiently transforming Escherichia coli called the "colony-to-lawn" method. The key steps are: 1) lysing plasmid-containing donor cells to release plasmids, 2) scraping recipient cells directly from a lawn on an agar plate into the lysate, allowing transformation without preparing competent cells. This method saves time compared to traditional methods by skipping plasmid purification and competent cell preparation. It was also used to conveniently screen positive clones after DNA ligation and transformation during construction of a mutant library.
- The study investigated the effect of calcium chloride concentration on the transformation efficiency of E. coli with plasmids pUC19 and pBR322, which differ in size.
- Maximum transformation efficiency was observed at 0.15M CaCl2 for pUC19 and 0.1M CaCl2 for the larger pBR322 plasmid.
- Increasing calcium chloride concentration above these levels decreased transformation efficiency for both plasmids, with no transformants observed above 0.2M, possibly due to decreased cell viability in hypertonic conditions.
This study investigates the localization and function of VDAC4, a porin protein in Tetrahymena thermophila mitochondria. Bioinformatics analysis predicted VDAC4 contains a conserved Porin3 domain found in mitochondrial porins and Tom40 proteins. The researcher amplified and cloned the VDAC4 gene, created a YFP fusion construct, transformed Tetrahymena cells, and observed YFP localization using microscopy. YFP localized to punctate structures consistent with mitochondrial localization, supporting VDAC4 involvement in mitochondrial membrane processes.
Transcription factors of the nuclear factor κ B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC50 for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z′ >0.95), speed, and robustness in a screen of a compound collection. It identified the IκB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocy- toplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor κ B
Differentiation & Activity Of Human Pre-Osteoclasts On Chitosan-Ashish Sh...as747
1. The document studied the effects of chitosan on human pre-osteoclast differentiation and activity. Pre-osteoclasts were cultured on cementek, cementek/chitosan, and PMMA pellets for 7 days.
2. Live/dead staining showed high viability on all materials. TRACP staining showed large, positive multinucleated osteoclasts on cementek but few on cementek/chitosan. Scanning electron microscopy and gene expression analysis were also performed.
3. The results suggest that chitosan may inhibit pre-osteoclast differentiation and reduce osteoclast activity, which could impact bone remodeling. Further analysis of chitosan's
1. The document describes methods for stably transfecting cell lines, including calcium phosphate transfection, lipofection, and spontaneous transfection.
2. It examines the efficiency of transfection for different cell lines using green fluorescent protein and red fluorescent protein plasmids.
3. Results show that calcium phosphate transfection was more efficient than spontaneous transfection for most cell lines tested, except HeLa cells. Stable transfection of HeLa cells over 21 days resulted in gradually increasing fluorescent intensity that then remained constant.
This document summarizes an experiment optimizing expression of G-protein coupled receptors (GPCRs) in HEK293 cells using a bioreactor. The researchers found that using a bioreactor allowed for higher cell densities and expression levels of over 1 mg of recombinant GPCR per liter of culture, compared to 0.1-0.2 mg/L using spinner flasks. A large-scale run in the bioreactor produced 10 mg of purified, bioactive recombinant GPCR. The bioreactor setup improved expression levels for structural characterization of this important class of membrane proteins.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
This document summarizes an experiment that aimed to change both the expression level and color of the fluorescent protein mCherry. The experiment involved:
1) Using restriction digestion and ligation to swap the promoter of mCherry from low to high expression, resulting in more mCherry colonies.
2) Attempting site-directed mutagenesis to change mCherry to mOrange but this was unsuccessful, as no orange colonies were observed.
3) Characterizing the fluorescence of mCherry, mOrange from a partner, and a negative control colony, finding mOrange emitted better at 500nm.
- The study investigated the effect of calcium chloride concentration on the transformation efficiency of E. coli with plasmids pUC19 and pBR322, which differ in size.
- Maximum transformation efficiency was observed at 0.15M CaCl2 for pUC19 and 0.1M CaCl2 for the larger pBR322 plasmid.
- Increasing calcium chloride concentration above these levels decreased transformation efficiency for both plasmids, with no transformants observed above 0.2M, possibly due to decreased cell viability in hypertonic conditions.
This study investigates the localization and function of VDAC4, a porin protein in Tetrahymena thermophila mitochondria. Bioinformatics analysis predicted VDAC4 contains a conserved Porin3 domain found in mitochondrial porins and Tom40 proteins. The researcher amplified and cloned the VDAC4 gene, created a YFP fusion construct, transformed Tetrahymena cells, and observed YFP localization using microscopy. YFP localized to punctate structures consistent with mitochondrial localization, supporting VDAC4 involvement in mitochondrial membrane processes.
Transcription factors of the nuclear factor κ B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC50 for the nuclear export blocker leptomycin B of 2.4 ng/mL (4.4 nM). The method demonstrated high selectivity (Z′ >0.95), speed, and robustness in a screen of a compound collection. It identified the IκB protein kinase inhibitor BAY 11 7082 as an import inhibitor, the p38 mitogen-activated protein (MAP) kinase inhibitor PD98509 as an import enhancer, and phorbol ester as an export inhibitor. The results establish a robust method for identifying compounds regulating nucleocy- toplasmic import or export and also implicate MAP kinases in nuclear import of nuclear factor κ B
Differentiation & Activity Of Human Pre-Osteoclasts On Chitosan-Ashish Sh...as747
1. The document studied the effects of chitosan on human pre-osteoclast differentiation and activity. Pre-osteoclasts were cultured on cementek, cementek/chitosan, and PMMA pellets for 7 days.
2. Live/dead staining showed high viability on all materials. TRACP staining showed large, positive multinucleated osteoclasts on cementek but few on cementek/chitosan. Scanning electron microscopy and gene expression analysis were also performed.
3. The results suggest that chitosan may inhibit pre-osteoclast differentiation and reduce osteoclast activity, which could impact bone remodeling. Further analysis of chitosan's
1. The document describes methods for stably transfecting cell lines, including calcium phosphate transfection, lipofection, and spontaneous transfection.
2. It examines the efficiency of transfection for different cell lines using green fluorescent protein and red fluorescent protein plasmids.
3. Results show that calcium phosphate transfection was more efficient than spontaneous transfection for most cell lines tested, except HeLa cells. Stable transfection of HeLa cells over 21 days resulted in gradually increasing fluorescent intensity that then remained constant.
This document summarizes an experiment optimizing expression of G-protein coupled receptors (GPCRs) in HEK293 cells using a bioreactor. The researchers found that using a bioreactor allowed for higher cell densities and expression levels of over 1 mg of recombinant GPCR per liter of culture, compared to 0.1-0.2 mg/L using spinner flasks. A large-scale run in the bioreactor produced 10 mg of purified, bioactive recombinant GPCR. The bioreactor setup improved expression levels for structural characterization of this important class of membrane proteins.
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
This document summarizes an experiment that aimed to change both the expression level and color of the fluorescent protein mCherry. The experiment involved:
1) Using restriction digestion and ligation to swap the promoter of mCherry from low to high expression, resulting in more mCherry colonies.
2) Attempting site-directed mutagenesis to change mCherry to mOrange but this was unsuccessful, as no orange colonies were observed.
3) Characterizing the fluorescence of mCherry, mOrange from a partner, and a negative control colony, finding mOrange emitted better at 500nm.
Genetically engineered E. coli were designed to express enhanced green fluorescent protein (EGFP). The EGFP gene was PCR amplified from a plasmid and inserted into the expression vector pET-41a. This recombinant DNA was transformed into E. coli. While some colonies were observed, none exhibited green fluorescence under UV light. Errors in PCR amplification and potential issues with the recombinant DNA inserts suggest the hypothesis that E. coli transformants would express EGFP was not supported.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This document describes a new method called DNA assembler that allows for the rapid assembly of entire biochemical pathways in a single step using in vivo homologous recombination in yeast. The method is demonstrated by assembling a 9 kb D-xylose utilization pathway (3 genes), an 11 kb zeaxanthin biosynthesis pathway (5 genes), and a 19 kb combined D-xylose and zeaxanthin pathway (8 genes), all with high efficiencies of 70-100%. DNA assembler represents an improvement over previous methods for pathway construction as it is faster, requires only simple DNA preparation and one-step yeast transformation, and can assemble larger pathways without limitations on restriction sites.
Molecular Cloning of the Structural Gene for ExopolygalacturonateAlan Brooks
This document summarizes research on the cloning and characterization of a gene (pelX) from Erwinia chrysanthemi that encodes an exopolygalacturonate lyase (ExoPL). The pelX gene was cloned from a mutant strain lacking known pectate lyase genes. ExoPL was purified from a recombinant E. coli strain and characterized. A pelX mutant was constructed in E. chrysanthemi but retained pathogenicity, indicating ExoPL does not contribute to tissue maceration ability.
The study aimed to test the specificity of two antibodies (033 and 1-9E10) for binding the c-myc protein. Human colon carcinoma cells were extracted to obtain cytosolic, nuclear, and nuclear pellet extracts. The 033 antibody bound c-myc in the cytosolic extract at approximately 110 kDa, but the 9E10 antibody did not bind c-myc. This suggests c-myc is not restricted to the nucleus and more research is needed to fully understand c-myc and its role in cancer.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
Transformation of saccharomyces cerevisiae and other fungiCAS0609
This document reviews methods for transforming various fungi, including Saccharomyces cerevisiae, with a focus on improvements since 2001. It summarizes the original spheroplast and lithium acetate methods for S. cerevisiae transformation, noting key steps and findings. The document then presents a proposed model for the mechanism of S. cerevisiae transformation based on recent reports, suggesting DNA attaches to the cell wall and enters via endocytosis, with polyethylene glycol playing a role in attachment and membrane effects.
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
This document summarizes a research study exploring how phosphorylation affects the organization of keratin 14 filaments during cell cycle progression. The researchers hypothesized that constant phosphorylation (phospho-mimic) or dephosphorylation (phospho-null) of keratin 14 would disrupt normal cell division. They used site-directed mutagenesis to create phospho-mimic and phospho-null mutants of keratin 14, then analyzed cell phenotype after transfection. Preliminary results showed the phospho-null S33A mutant had more keratin bridges than wild type, suggesting phosphorylation at this site affects filament reorganization during cell division.
Chloroplasts contain their own DNA and are the site of photosynthesis. Chloroplast transformation involves delivering a vector with the gene of interest and a selectable marker flanked by chloroplast DNA sequences for homologous recombination. The vector is delivered using biolistics or PEG-mediated transformation. Transformed cells are selected using antibiotic resistance and regenerated into plants. Chloroplast transformation allows high-level expression of transgenes due to high copy number and avoids gene silencing.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Plastid transformation allows for high levels of protein expression and improved biosafety compared to nuclear transformation. Plastids are organelles that perform important functions in plant cells, including photosynthesis, and contain their own circular genome. Transformation involves delivering foreign DNA to plastids using biolistics or PEG-mediated transformation, then selecting for integration into the plastid genome by homologous recombination. Successful plastid transformation has been achieved in many plant species and used to confer valuable agronomic traits and produce human therapeutic proteins.
This study developed a new fluorescence-based assay to quantify endosome fusion in living cells. The assay uses BODIPY-labeled avidin, which exhibits a 10-fold increase in fluorescence upon binding to biotin. BHK fibroblasts were pulse-labeled with BODIPY-avidin and a red fluorescent marker. After specified chase times, a second cohort of endosomes was pulse-labeled with biotin-conjugated probes. Fusion was detected by increased BODIPY fluorescence in individual endosomes, measured by ratio imaging. Applying this assay, the study found that over 90% of avidin-labeled endosomes fused within 10 minutes, with fusion decreasing at longer chase times, indicating endosome
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
A rapid and efficient assay for extracting dna from fungiCAS0609
The document describes a new rapid method for extracting DNA from fungi in a batch format. The method involves scraping fungal tissue, subjecting it to seven rounds of freeze/thaw lysis using dry ice and boiling water, followed by boiling for 30 minutes and grinding. The Qiagen DNeasy Plant Tissue Kit is then used to purify the extracted DNA. Testing showed this new method allowed effective DNA extraction from multiple fungal isolates simultaneously in a simple and reliable way. It provides researchers an improved technique for obtaining DNA from fungi for molecular assays compared to more specialized or time-consuming existing methods.
Genetically engineered E. coli were designed to express enhanced green fluorescent protein (EGFP). The EGFP gene was PCR amplified from a plasmid and inserted into the expression vector pET-41a. This recombinant DNA was transformed into E. coli. While some colonies were observed, none exhibited green fluorescence under UV light. Errors in PCR amplification and potential issues with the recombinant DNA inserts suggest the hypothesis that E. coli transformants would express EGFP was not supported.
Efficient transformation of lactococcus lactis il1403 and generation of knock...CAS0609
This document describes an optimized protocol for efficiently transforming Lactococcus lactis IL1403 by electroporation. Key aspects of the protocol include growing cells in media supplemented with glycine and sucrose, harvesting them at mid-late log phase, washing them in buffers containing sucrose and EDTA, and electroporating them with a resistor in series. The utility of the protocol was demonstrated by generating single and double gene knock-out mutants using non-replicating vectors. Transformation efficiencies as high as 106 cfu/μg of DNA were achieved, allowing for genetic manipulation of L. lactis IL1403.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This document describes a new method called DNA assembler that allows for the rapid assembly of entire biochemical pathways in a single step using in vivo homologous recombination in yeast. The method is demonstrated by assembling a 9 kb D-xylose utilization pathway (3 genes), an 11 kb zeaxanthin biosynthesis pathway (5 genes), and a 19 kb combined D-xylose and zeaxanthin pathway (8 genes), all with high efficiencies of 70-100%. DNA assembler represents an improvement over previous methods for pathway construction as it is faster, requires only simple DNA preparation and one-step yeast transformation, and can assemble larger pathways without limitations on restriction sites.
Molecular Cloning of the Structural Gene for ExopolygalacturonateAlan Brooks
This document summarizes research on the cloning and characterization of a gene (pelX) from Erwinia chrysanthemi that encodes an exopolygalacturonate lyase (ExoPL). The pelX gene was cloned from a mutant strain lacking known pectate lyase genes. ExoPL was purified from a recombinant E. coli strain and characterized. A pelX mutant was constructed in E. chrysanthemi but retained pathogenicity, indicating ExoPL does not contribute to tissue maceration ability.
The study aimed to test the specificity of two antibodies (033 and 1-9E10) for binding the c-myc protein. Human colon carcinoma cells were extracted to obtain cytosolic, nuclear, and nuclear pellet extracts. The 033 antibody bound c-myc in the cytosolic extract at approximately 110 kDa, but the 9E10 antibody did not bind c-myc. This suggests c-myc is not restricted to the nucleus and more research is needed to fully understand c-myc and its role in cancer.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
Generation of MRP2 Efflux Transporter Knock-Out in HepaRG Cell Linemdmitc
The document describes experiments characterizing a HepaRG cell line with MRP2 (an efflux transporter) knocked out using zinc finger nuclease technology. Western blot and immunostaining showed loss of MRP2 protein expression in knockout cells. Assays found control cells accumulated the fluorescent MRP2 substrate CDCF in bile canaliculi, while knockout cells did not, demonstrating functional loss of MRP2. The MRP2 knockout cell line allows investigation of MRP2-associated drug toxicity without its protective efflux.
Transformation of saccharomyces cerevisiae and other fungiCAS0609
This document reviews methods for transforming various fungi, including Saccharomyces cerevisiae, with a focus on improvements since 2001. It summarizes the original spheroplast and lithium acetate methods for S. cerevisiae transformation, noting key steps and findings. The document then presents a proposed model for the mechanism of S. cerevisiae transformation based on recent reports, suggesting DNA attaches to the cell wall and enters via endocytosis, with polyethylene glycol playing a role in attachment and membrane effects.
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
This document summarizes a research study exploring how phosphorylation affects the organization of keratin 14 filaments during cell cycle progression. The researchers hypothesized that constant phosphorylation (phospho-mimic) or dephosphorylation (phospho-null) of keratin 14 would disrupt normal cell division. They used site-directed mutagenesis to create phospho-mimic and phospho-null mutants of keratin 14, then analyzed cell phenotype after transfection. Preliminary results showed the phospho-null S33A mutant had more keratin bridges than wild type, suggesting phosphorylation at this site affects filament reorganization during cell division.
Chloroplasts contain their own DNA and are the site of photosynthesis. Chloroplast transformation involves delivering a vector with the gene of interest and a selectable marker flanked by chloroplast DNA sequences for homologous recombination. The vector is delivered using biolistics or PEG-mediated transformation. Transformed cells are selected using antibiotic resistance and regenerated into plants. Chloroplast transformation allows high-level expression of transgenes due to high copy number and avoids gene silencing.
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellula...mdmitc
MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.
Plastid transformation allows for high levels of protein expression and improved biosafety compared to nuclear transformation. Plastids are organelles that perform important functions in plant cells, including photosynthesis, and contain their own circular genome. Transformation involves delivering foreign DNA to plastids using biolistics or PEG-mediated transformation, then selecting for integration into the plastid genome by homologous recombination. Successful plastid transformation has been achieved in many plant species and used to confer valuable agronomic traits and produce human therapeutic proteins.
This study developed a new fluorescence-based assay to quantify endosome fusion in living cells. The assay uses BODIPY-labeled avidin, which exhibits a 10-fold increase in fluorescence upon binding to biotin. BHK fibroblasts were pulse-labeled with BODIPY-avidin and a red fluorescent marker. After specified chase times, a second cohort of endosomes was pulse-labeled with biotin-conjugated probes. Fusion was detected by increased BODIPY fluorescence in individual endosomes, measured by ratio imaging. Applying this assay, the study found that over 90% of avidin-labeled endosomes fused within 10 minutes, with fusion decreasing at longer chase times, indicating endosome
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
A rapid and efficient assay for extracting dna from fungiCAS0609
The document describes a new rapid method for extracting DNA from fungi in a batch format. The method involves scraping fungal tissue, subjecting it to seven rounds of freeze/thaw lysis using dry ice and boiling water, followed by boiling for 30 minutes and grinding. The Qiagen DNeasy Plant Tissue Kit is then used to purify the extracted DNA. Testing showed this new method allowed effective DNA extraction from multiple fungal isolates simultaneously in a simple and reliable way. It provides researchers an improved technique for obtaining DNA from fungi for molecular assays compared to more specialized or time-consuming existing methods.
An improved method for genomic dna extraction from strawberry leavesCAS0609
This document describes an improved method for extracting genomic DNA from strawberry leaves. The standard CTAB method was modified by adding two additional steps: 1) lyophilizing the leaf tissue overnight to remove water content, and 2) macerating the dried tissue in the presence of polyvinylpyrrolidone (PVP) to prevent DNA binding with polyphenols. Results showed the modified method produced DNA of higher quality and quantity compared to the standard method, indicating lyophilization and PVP improve strawberry leaf DNA extraction. On average, the modified method yielded 57.3% more DNA than the standard method. The modified method also produced less oxidized DNA pellets, showing its effectiveness in preventing polyphenol
Professor Oak is conducting research on the effects of temperature changes on Pokemon and needs temperature data from Pallet Town. The diurnal temperature range in Pallet Town on January 6, 2013 was 15.4°C. He also needs the mean monthly temperature for February 2010 in Pallet Town, which is calculated to be 15.2°C based on the daily temperatures provided.
The known unknowns of antigen processing and presentationCAS0609
1) The pathways of antigen processing and presentation by MHC class I and class II molecules are generally well understood but some aspects remain unclear or controversial.
2) Dendritic cells play a key role in antigen presentation due to their ability to undergo maturation in response to pathogens, regulating antigen acquisition and presentation.
3) Recent evidence suggests additional cellular pathways, such as autophagy, contribute to antigen processing beyond just the proteasome and lysosomal proteases, but their specific roles require further study.
This document provides a protocol for extracting ancient DNA from bones and teeth. The method aims to maximize DNA recovery while minimizing co-extraction of PCR inhibitors. Key steps include extracting DNA from bone powder using an EDTA-proteinase K buffer, then purifying the DNA by binding it to silica in the presence of guanidinium thiocyanate. All steps are done at room temperature to reduce DNA degradation. The protocol yields DNA extracts within 2 days and has advantages over other methods such as being quick, scalable, simple to implement, and efficiently removing PCR inhibitors.
Glass bead transformation method for gram positive bacteriaCAS0609
This study developed a simple glass bead transformation method for introducing DNA into Gram-positive bacteria. The method involves treating bacterial protoplasts with glass beads, DNA, and polyethylene glycol. Using this method, the plasmid pGK12 was successfully introduced into several Gram-positive bacteria, including Enterococcus faecalis, Lactobacillus casei, Lactococcus lactis, Leuconostoc dextranicum, Listeria innocua, Staphylococcus aureus, and Streptococcus pneumoniae. Transformation frequencies ranged from 3.56 x 103 to 6.62 x 103 colonies per microgram of pGK12. This glass bead method provides an inexpensive and reproducible way to transform Gram
Dna extraction from fresh or frozen tissuesCAS0609
This document provides a protocol for extracting DNA from fresh or frozen tissues. The protocol involves disaggregating tissue samples and lysing cells to release DNA. Proteins are then digested and DNA is separated from other cellular components using phenol/chloroform extraction. The extracted DNA is precipitated and purified by ethanol precipitation. DNA concentration and quality can be assessed using spectrophotometry and gel electrophoresis. The protocol notes that traditional organic extraction effectively isolates high molecular weight DNA but commercial non-organic kits provide a faster alternative while avoiding toxic phenol.
Tugas 1 (kerangka & jaring2 bangun ruang )dhena175
Dokumen tersebut membahas tentang standar kompetensi dan indikator pembelajaran mengenai bangun ruang datar dan bangun ruang, yang mencakup kubus, balok, prisma, dan limas. Dokumen tersebut juga berisi contoh soal latihan mengenai membuat jaring-jaring dan mengidentifikasi bangun ruang datar dan bangun ruang.
This document contains links to YouTube videos about different types of livestock farming and mining in Spain, including bovine, ovine, caprine, porcine, avian, equine farming as well as beekeeping, mining, and fishing. It provides online resources for learning more about various agricultural and extraction industries through video format.
Biolostic transformation of a procaryote, bacillus megateriumCAS0609
This document describes a new method for transforming the bacterium Bacillus megaterium using biolistic transformation. Key findings include:
1) Plasmid DNA was coated onto tungsten microprojectiles and accelerated into B. megaterium cells using a helium-driven biolistic device.
2) Over 104 transformants per treated plate were obtained after optimizing biological and physical parameters of the biolistic process.
3) All strains of B. megaterium tested were successfully transformed, though efficiency varied between strains. This is the first report of biolistic transformation of a prokaryote.
Genomic dna from different biological materialsCAS0609
This document describes methods for extracting high-quality genomic DNA from different biological materials, including Gram-positive and Gram-negative bacteria and fungal mycelium and spores. It provides detailed protocols and lists the necessary materials for extracting genomic DNA from these sources using methods such as CTAB, phenol-chloroform, and commercial kits. The goal is to describe optimized procedures for efficiently extracting genomic DNA suitable for downstream applications like PCR and library cloning.
Dna extraction from blood and forensic samplesCAS0609
This document provides instructions for extracting DNA from various forensic samples, including blood, absorbing substrates like cloth or paper, and non-absorbing substrates like metal or plastic. The summary is as follows:
1) Precautions must be taken when handling forensic samples to prevent contamination, including working in a dedicated clean room, using dedicated equipment and reagents, frequent changing of gloves and cleaning of surfaces.
2) DNA can be extracted from blood samples by lysing red blood cells, then purifying the DNA through phenol-chloroform extraction and ethanol precipitation.
3) For absorbing substrates like cloth or paper, a small piece is cut and placed in lysis buffer for extraction. For non-absorbing substrates,
This document describes methods for stably transfecting cell lines to create new cell lines. It compares the calcium phosphate precipitation method, spontaneous transfection method, and lipofection method. It also details monitoring transfection efficiency by fluorescence microscopy and spectroscopy. Results show the calcium phosphate method worked best for most cell lines tested except HeLa cells. Stable transfection of HeLa cells with a plasmid increased fluorescence over 21 days then plateaued. Plasmid DNA concentration did not correlate with fluorescence yield.
This document describes methods for stably transfecting cell lines to create new cell lines. It compares the calcium phosphate precipitation method, spontaneous transfection method, and lipofection method. It also details monitoring transfection efficiency by fluorescence microscopy and spectroscopy. Results show the calcium phosphate method worked best for most cell lines tested except HeLa cells. Stable transfection of HeLa cells with a plasmid increased fluorescence over 21 days then plateaued. Plasmid DNA concentration did not correlate with fluorescence yield.
High efficiency 5 min transformation of escherichia coliCAS0609
This document describes a new method for transforming E. coli cells that takes only 5 minutes, compared to the standard 1.5 hour protocol. Key findings include:
1) Incubating cells with DNA on ice for 1-180 minutes before spreading directly onto pre-warmed plates at 37°C resulted in up to double the transformation efficiency compared to the standard heat shock method.
2) For most antibiotic resistance markers, there was no advantage to the standard 30-60 minute recovery period at 37°C after heat shock - the direct spreading method worked as well.
3) The new 5 minute method produced similar high transformation rates for a variety of plasmid and cell line combinations tested.
This document summarizes a study on the heterologous expression and characterization of the c1 dioxygenase enzyme from Tetranychus urticae. Key points:
- The c1 dioxygenase gene was inserted into a plasmid and transformed into E. coli cells for expression. However, initial expression attempts did not show overexpression of the protein.
- Additional expression trials were conducted by varying culture conditions and bacterial clones. SDS-PAGE analysis showed some potential overexpression in new clones induced overnight at 37°C, though further optimization is still needed.
- Once expression is optimized, the goal is to purify the recombinant protein using its His-tag and proceed with structural characterization through crystallization,
An Understanding Of Bacterial Transformation By Plasmid DnaGina Buck
Bacterial plasmids are small, circular DNA molecules within bacteria that are separate from the bacterial chromosome. Plasmids can contain genes that provide bacteria with useful traits like antibiotic resistance. During genetic transformation, the plasmid is introduced to recipient bacteria where it can be replicated independently of the bacterial chromosome. The foreign DNA from the plasmid is then expressed in the recipient bacteria, altering its genotype and phenotype. This allows bacteria to horizontally acquire new genes from plasmids and gain traits like antibiotic resistance without direct contact between bacterial cells.
This paper describes a novel method for generating large libraries for directed evolution of proteins using error-prone PCR and a Kunkel-like mutagenesis reaction. The method uses error-prone PCR to generate a mutated DNA fragment, with one primer containing phosphorothioate modifications. Treatment with exonuclease preferentially removes the non-modified strand, generating a single-stranded "megaprimer". This megaprimer is then used in a Kunkel-like reaction with a uracilated template to introduce mutations efficiently without subcloning. This approach enables generation of libraries with over 108 clones from a single E. coli transformation, which is more efficient than conventional error-prone PCR
Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. Mammalian cell culture technology has become a major field in modern biotechnology; mammalian cell culture refers to the cells of a mammalian, isolated from specific tissues (i.e. skin, liver, glands, etc.) and further cultivated and reproduced in an artificial medium. Cell culture technology is currently playing a major role in toxicity testing, cancer research, virology, genetic engineering, and gene therapy.
OBJECTIVE:
To observe the transfection of CHO and HEK cells with GFP
To observe the recombinant GFP using Western Blotting
To purify the transfected HEK and CHO cells using AKTA Pure Purification
The document describes the process for constructing a deletion strain library for Thermococcus kodakarensis. It involves first cloning each of the 2,306 open reading frames (ORFs) from the T. kodakarensis genome into plasmids to generate "A-plasmids". Then, inverse PCR is used to delete the target gene from each A-plasmid, generating "B-plasmids". The B-plasmids are then used to transform T. kodakarensis cells to introduce the deletions into the genome via homologous recombination, creating a library of deletion strains with individual ORFs removed. The status of the project is that 87% of
The document reports on DNA and protein techniques practicals performed by Kariuki Samuel Mundia. It describes cloning the nanobody gene using restriction digestion of the nanobody PCR fragment and PHEN6c plasmid with Eco911 and PstI enzymes. The DNA fragments were purified and ligated together. It also details plasmid isolation from E. coli cells and generating calcium chloride competent E. coli cells for heat shock transformation of the ligation mixture. The goal was to amplify the nanobody gene by inserting it into the PHEN6c plasmid vector and transforming it into E. coli cells.
Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
The document discusses various artificial transformation methods for improving the efficiency of plasmid DNA transformation in bacteria. It describes chemical transformation using calcium chloride, electroporation using electric pulses, physical transformation using nanomaterials and friction, and combined transformation methods that integrate multiple approaches. The conclusion states that while artificial methods increase transformation effectiveness over natural processes, simpler and more universal techniques are still needed that can transform a wide range of bacterial species.
This document summarizes a plasmid lab report that used pUC19 plasmids as the vector for E. coli transformation due to its small size, high uptake efficiency, and fast replication time. Key features of pUC19 include an origin of replication and multiple cloning sites. Transformed E. coli were able to grow on agar plates containing ampicillin due to the plasmid containing an ampicillin resistance gene. Non-recombinant E. coli colonies were blue due to expression of the lacZ gene, while recombinant colonies were white due to insertion of the CIH-1 gene within the multiple cloning sites.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
You have identified a mutation in E. coli K-12 that causes it to bec.pdfvenkatesh24685
You have identified a mutation in E. coli K-12 that causes it to become sensitive to the the
antibiotic azithromycin (azi). You sequence the chromosome of the aziS strain and localize the
mutation to genX and near the cysABC operon that encodes the only genes required for cysteine
biosynthesis (see diagram). In order to study the impact of the genX* mutation in a pathogenic
strain, you would like to move this allele into a uropathogenic E. coli (UPEC) strain background.
Design a strategy to move your genX* mutation from E. coli K-12 into the UPEC strain using
the strains and plasmids described below. Use point form to describe your strategy. Include a
description of all conditions used for selections and/or screens and be clear, but brief about how
you will carry out each step. Hint: your answer will require the use of transformation,
transposition, and transduction and should include a protocol for a genetic screen for a
transposon mutant & the use of generalized transduction to move alleles between strains (9
marks): •Suicide plasmid pMUT: plasmid that encodes the transposon Tn5 & ampicillin
resistance; can only replicate at 30oC, not 37oC. the Tn5 element contains a gene that confers a
kanamycin resistant phenotype (KnR) •E. coli K-12 genX* : E. coli K-12 strain carrying your
genX* mutation that confers azithromycin sensitivity •UPEC wild-type strain •Generalized
transducing phage P1, which can infect both E. coli K-12 and UPEC
(all information is given: need to design an experiment with non-mobilizable/ suicide vectors/
transposons/ selection process etc). UPEC chromosome genX cySA cysB *cySA, B, Care only
genes required for biosynthesis of cysteine EC Ampicillin Ori pMUT Tn5 (KnR
Solution
1. Transduction and Experimental steps for transduction:-
Transduction is the heritable transfer of bacterial DNA from one cell (donor, here it is E Coli
K12) to another (recipient, EColi, UPEC) by a bacteriophage. Tansducing bacteriophage
particles are formed in donor bacterial cells during phage development. Generalized transducing
phage particles carry a random fragment of host chromosomal DNA approximately the same
length as P1 DNA. The phage particles completely lack DNA originating from the phage
genome and contain instead oly Bacterila DNA sequesnces. New genotypes in the recipient cells
result from homoogous recombination, which can lead to the replacement of a recipient gene by
an allele acquired from the donor genome via the transducing phage.
a. Ecoli K 12 (Host cell) infected by phage
b. Host cell DNA broken down into smaller pieces and proteins and phage DNA synthesized
c. Bacterial host DNA is packaged in some viral capsids that are released through lysis of
bacterial cell.
d. Transducing phage with host DNA infest new recipient cell.
e. Recombinant new cell contain mixture of donor DNA and its own DNA.
2. Transformation:- It is genetic alteration of a cell resulting from the direct uptake and
incorporation of exogenous genetic material fr.
for cloning and expression of exogenous gene or gene throthrough vector it must be introduced into the host cell through transformation , ,transduction, electroporation gene gun etc.
This document discusses various gene transfer methods in animals, including viral and non-viral approaches. Viral methods include retroviruses, adenoviruses, adeno-associated viruses, baculoviruses, which can integrate the transferred gene into the host cell's genome. Non-viral methods discussed are transfection techniques like calcium phosphate precipitation, DEAE-dextran, lipids, microinjection, gene guns, ultrasound, and electroporation. These physical methods deposit naked DNA directly into cells without viral vectors. The document provides detailed protocols for many of these gene transfer techniques.
This document summarizes a seminar on using Caco-2 monolayers to study transport across the intestinal barrier. The Caco-2 cell line spontaneously differentiates into enterocytes that form a polarized monolayer mimicking intestinal absorption. Key aspects covered include Caco-2 cell culture and characterization, permeability assays to measure transport, and applications in drug development. Validation is done using reference compounds to classify drug absorption. Considerations for biological factors and analytical methods are also discussed.
Genetic transformation & success of DNA ligation Sabahat Ali
DNA is ligated through DNA Ligase, problems may occur during DNA ligation are
1) vector cyclization
2) vector-vector concatemers
3) target DNA-target DNA ligation
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A colony to-lawn method for efficient transformation of escherichia coli
1. Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
A colony-to-lawn method for efficient transformation of
Escherichia coli
Y. An, A. Lv and W. Wu
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China
Keywords Abstract
chemical transformation, competent cells,
electroporation, low-copy-number plasmid, Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli
mutant library. transformation method for changing hosts of plasmids, which can also facilitate
the selection of positive clones after DNA ligation and transformation.
Correspondence Methods and Results: A single fresh colony from plasmid-containing donor
Wenfang Wu, Institute of Applied Ecology,
strain is picked up and suspended in 75% ethanol. Cells are pelleted and resus-
Chinese Academy of Sciences. No.72 Wenhua
Road. Shenyang 110016, China.
pended in CaCl2 solution and lysed by repetitive freeze–thaw cycles to obtain
E-mail: wshr100@sina.com.cn plasmid-containing cell lysate. The E. coli recipient cells are scraped from the
lawn of LB plate and directly suspended in the plasmid-containing cell lysate
2010 ⁄ 0411: received 11 March 2010, revised for transformation. Additionally, a process based on colony-to-lawn transfor-
4 April 2010 and accepted 21 April 2010 mation and protein expression was designed and conveniently used to screen
positive clones after DNA ligation and transformation.
doi:10.1111/j.1472-765X.2010.02864.x
Conclusions: With this method, a single colony from plasmid-containing
donor strain can be directly used to transform recipient cells scraped from
lawn of LB plate. Additionally, in combination with this method, screening of
positive clones after DNA ligation and transformation can be convenient and
time-saving.
Significance and Impact of the Study: Compared with current methods, this
procedure saves the steps of plasmid extraction and competent cell preparation.
Therefore, the method should be highly valuable especially for high-throughput
changing hosts of plasmids during mutant library creation.
efficiency (Okamoto et al. 1997; McCormac et al. 1998).
Introduction
In addition, a liposome-mediated transformation system
Changing hosts of plasmids by transformation is essential has been developed, because bacterial cells were found to
for many experiments in molecular biology, molecular be susceptible to transformation by liposomes (Kawata
genetics, etc. The CaCl2-mediated chemical transforma- et al. 2003). Although the methods described earlier have
tion is one of the most commonly used transformation provided various choices for efficient transformation of
methods until now. With this method, after treatment Escherichia coli, they are all dependent on the extraction
with CaCl2, a transient state of ‘competence’ is introduced of plasmid DNA beforehand. Therefore, when changing
to the recipient cells, and the cells are more likely to the hosts of hundreds or thousands of plasmids is
incorporate bacteriophage DNA or plasmid DNA (Man- performed, the work should be very time-consuming,
del and Higa 1970; Cohen et al. 1972; Oishi and Cosloy expensive and inconvenient.
1972). Some modified methods have been designed to In addition, during molecular cloning or construction
promote the efficiency of chemical transformation (Golub of mutant libaries, frameshift mutations often occur,
1988; Liu and Rashidbaigi 1990; Tang et al. 1994; Pope which may prevent the expression of proper proteins in
and Kent 1996; Chen et al. 2001; Zeng et al. 2006). E. coli. Although these mutations can be detected and
Another efficient transformation method is electro- removed by DNA sequencing of randomly selected clones,
poration, which can introduce a higher transformation the process is inconvenient especially when changing
ª 2010 The Authors
98 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103
2. Y. An et al. How to make transformation more efficient
hosts of multiple plasmids are performed during muta- BL21(DE3) harbouring pETM11-P450-BM3 obtained
tion library creation. To address these problems, we either from colony-to-lawn transformation or from chem-
describe a rapid, convenient and inexpensive method for ical transformation were cultured in TB media supple-
changing E. coli hosts of plasmids. Additionally, based on mented with kanamycin. The cultures were induced using
this method and protein expression, a process was IPTG (0Æ2 mmol l)1) at the exponential growth phase and
designed and conveniently used to screen positive clones incubated at 20°C with shaking at 150 rev min)1 over-
after DNA ligation and transformation. night. As a control, two colonies from the E. coli JM109
strain harbouring pETM11-P450-BM3 were also used for
induced protein expression as described earlier. Cells
Materials and methods
from these cultures were pelleted by centrifugation and
Escherichia coli JM109 strains harbouring plasmids pUC19 checked the expression levels of protein P450-BM3 by
(Ampr), pBR322 (Ampr), pYES2 (Ampr), pLysS (Camr), SDS-PAGE.
pSE380 (Ampr), pETM-11 (Kanr) and pETM11-p450BM3 A mutant library of P450-BM3 was generated by error-
(Kanr) were grown on antibiotic-supplemented LB agar prone PCR. The primers P450-For (5¢-GAGGGATACCA-
plates for 36 h. The concentrations of the antibiotics TGGCAATTAAAGAAATGCCTCAGCC-3¢) and P450-Rev
ampicillin, chloramphenicol and kanamycin were 50, 30 (5¢-CTCGCGGCCGCTTACCCAGCCCACACGTCTTTTG-
and 50 mg l)1 respectively. For each strain, a single CG-3¢) were used for PCR amplification. The PCR was
colony was carefully picked up without gouging the agar. performed in mixture containing 2 ng of P450-BM3 tem-
Each colony was suspended in a tube containing 200 ll plate DNA, 0Æ5 lmol l)1 both primers, 1 mmol l)1
Milli-Q water followed by the addition of 600 ll ethanol d(C ⁄ T)TP, 0Æ2 mmol l)1 d(A ⁄ G)TP, 40 nmol l)1 MgCl2,
to the tube. The mixtures were put in room temperature 1· Taq polymerase buffer and 3 Unit Taq polymerase
for 5 min, and then the cells were pelleted by centrifuga- with a total volume of 50 ll. This reaction mixture was
tion. The tubes were put upside down for 10 min at heated at 95°C for 2 min followed by 30 cycles of incuba-
room temperature to dry pellets, and a 30-ll aliquot of tion at 95°C for 1 min, 48°C for 40 s, and 72°C for
0Æ1 mol l)1 CaCl2 was added to each tube and mixed 5 min and a final incubation at 72°C for 10 min. After
carefully. Then, the cells of different strains were lysed by purification, the PCR product was digested with NotI and
frozen at )80°C and thawed at 100°C for three cycles to NcoI and cloned into the corresponding restriction
obtain plasmid-containing cell lysates. The recipient strain enzyme sites of pETM11 vector and transformed into
BL21(DE3) was intensively grown on LB agar plates for E. coli JM109. Ten randomly selected transformants were
24 h to form lawn. The cells from lawn were carefully used to transform E. coli recipient strain BL21(DE3) with
scraped without gouging the agar and resuspended in five the colony-to-lawn transformation method. After trans-
times volume of ice-cold water. A 30-ll aliquot of cells formation, transformed bacteria were grown in 50-ml
suspension was transferred to each tube containing the auto-inducing media (ZYM-5052) (Studier 2005). The
plasmid-containing cell lysate and mixed gently. The mix- cultures were first incubated at 37°C till OD600 = 1 and
tures were incubated on ice for 15 min followed by heat then incubated at 20°C overnight with shaking at
shock at 42°C for 40 s to perform transformation. Trans- 150 rev min)1. Cells from 5 ml of each culture were pel-
formed bacteria were grown and selected by standard leted by centrifugation and used to check protein expres-
methods. The number of transformants after each trans- sion by SDS-PAGE, and the remaining cultures (about
formation with a single colony of plasmid-containing 45 ml for each) were kept at 4°C. The plasmids were
donor strain was calculated after incubation at 37°C for extracted from the remaining cultures of positive clones
24 h. After each transformation, the plasmids were with expected protein expression, and DNA sequencing
extracted from five randomly selected transformants and was performed.
re-transformed into BL21(DE3) competent cells with the
traditional chemical transformation method. This was
Results
used to check whether the antibiotic-resistant colonies
were real transformants or just E. coli mutants or contam- The colony-to-lawn transformation method for changing
inants. As a control, the cell lysates were directly spread hosts of plasmids is illustrated in Fig. 1a. The first step is
on antibiotic-supplemented LB agar plates to check preparation of plasmid-containing cell lysate. A single
whether all the cells were sterilized after 75% ethanol colony from plasmid donor strain is suspended in 75%
incubation and freeze–thaw cycles. Changing hosts of ethanol followed by centrifugation to get pellet, and then
plasmid pETM11-P450-BM3 from JM109 to BL21(DE3) the cells are resuspended in CaCl2 solution and lysed
was also performed with chemical transformation after by freeze–thaw cycles to obtain plasmid-containing cell
plasmid extraction. Then, two transformants of lysate. The second step is preparation of recipient cells for
ª 2010 The Authors
Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 99
3. How to make transformation more efficient Y. An et al.
(a) (b) Colonies from Colonies from
Colonies from plasmid-containing
plasmid-containing plasmid-containing Lawn from
donor strain donor strain recipient strain
donor strain
Lawn from
recipient strain
A single colony Liquid
culture
200 µl water Cells pelleted by Cells scraped
600 µl ethanol centrifugation from lawn
Water Suspension Competent cell
preparation
30 µl CaCI2 Freeze-thaw
solution cycles Plasmid
extraction
Transformation Transformation
Transformation
Chemical transformation Colony-to-lawn transformation
(c) (d)
M 1 2 3 120
Transformation frequencies
100
120- 80
100-
85- 60
40
50-
20
(kDa) 0
pUC19 pBR322 pYES2 pLysS pSE380 pETM-11 pETM 11-
p450BM3
Plasmids
Figure 1 The colony-to-lawn transformation method used for changing hosts of plasmid. (a) Outline of the colony-to-lawn transformation
method. A single colony from plasmid donor strain is washed with 75% ethanol and air-dried, and then cells are suspended in CaCl2 solution and
lysed by freeze–thaw cycles to obtain plasmid-containing cell lysate. At the same time, cells of plasmid recipient strain are scraped carefully from
fresh lawn and suspended in ice-cold water. Then, the recipient cells and plasmid-containing cell lysate are mixed gently and performed transfor-
mation by heat shock method. The transformed bacteria are grown and selected by standard methods. (b) Comparison of the colony-to-lawn
transformation method and the chemical transformation method. Plasmid extraction and competent cell preparation are essential steps for chemi-
cal transformation, but not necessary for colony-to-lawn transformation. (c) SDS-PAGE gel shows protein expression of P450-BM3 before and
after changing hosts of pETM11-P450-BM3 either by colony-to-lawn transformation or by chemical transformation. Lane M: protein molecular
weight marker; lane 1, after host changing of pETM11-P450-BM3 with the chemical transformation method; lanes 2, after host changing of
pETM11-P450-BM3 with the colony-to-lawn transformation method; lanes 3, before host changing of pETM11-P450-BM3 (i.e. protein expressed
in Escherichia coli JM109). (d) The numbers of transformants obtained by changing hosts of various plasmids with the colony-to-lawn transforma-
tion method. Each value represents the mean of five independent experiments.
transformation. Cells of E. coli recipient strain are scraped plasmid extraction and competent cell preparation steps
carefully from fresh lawn without gouging the agar and are needed (Fig. 1b). Using pETM11-P450-BM3 as a sam-
then suspended in ice-cold water. The third step is trans- ple, we changed its hosts from E. coli JM109 to
formation. An aliquot of recipient cells and plasmid-con- BL21(DE3) either by colony-to-lawn transformation or by
taining cell lysate are mixed gently and we performed chemical transformation. After IPTG induction, the simi-
transformation by heat shock method. Then, transformed lar expression levels of P450-BM3 protein were obtained
bacteria are grown and selected by standard methods. (Fig. 1c), indicating that there is no fundamental differ-
The colony-to-lawn transformation method is more ence between these transformants. We tested the colony-
convenient and rapid than current methods, because no to-lawn transformation method by using it to change the
ª 2010 The Authors
100 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103
4. Y. An et al. How to make transformation more efficient
hosts of various plasmids, including the low-copy-number containing cell lysate, indicating that 75% ethanol incuba-
plasmid pLysS. As a result, no less than 60 transformants tion and freeze–thaw cycles were efficient for sterilization,
were available after each transformation with a single col- and no transformants obtained after transformation were
ony of plasmid-containing donor strain (Fig. 1d). Addi- mutants or contaminants.
tionally, the method is very convenient, because the LB A process based on colony-to-lawn transformation and
agar plates with colonies of donor strains and recipient protein expression was designed and conveniently used to
strain can be stocked at 4°C for at least 7 days without remove frameshift mutations during the construction of
affecting the transformation obviously (data not shown). mutant library (Fig. 2a). Recombinant plasmids are
As a control, plasmids from the randomly selected trans- constructed and transformed into E. coli cloning strain,
formants were successfully re-transformed into E. coli followed by changing the hosts of plasmids from cloning
BL21(DE3) by chemical transformation, indicating that strain to expression strain with the colony-to-lawn trans-
the antibiotic-resistant colonies after colony-based trans- formation method. Then, randomly selected transfor-
formation were real transformants but not E. coli mutants mants are cultured in auto-inducing media overnight. An
or contaminants. In addition, no colony was found on the aliquot of each culture is used to check protein expression
antibiotic-containing agar plates spread with the plasmid- by SDS-PAGE, and only the positive clones having
(a) Construction of (b) Construction of
recombinant plasmid recombinant plasmid
Transformation
Transformation
Colonies of
Colonies of
plasmid-containing plasmid-containing
donor strain donor strain
Colony-based Overnight cultures
transformation derived from
One day single colonies
1 2 3 4
Liquid culture of Less than
recipient cells two days
1 2 3 4 1 3 Extract plasmid from
each clone
2 4
Transformation into
1 2 3 4 recipient cells
1
3 Induced expression
Extract plasmids from 1 2 3 4
positive clones SDS-Page analysis One day
1 2 3 4
DNA Sequencing or
functional analysis SDS-Page
analysis
DNA Sequencing or
functional analysis
Figure 2 Protein expression in combination with the colony-to-lawn transformation method to screen in-frame clones from mutant library. (a)
Outline of the experimental strategy. Plasmids from mutant library construction were changed hosts from cloning strain to expression strain with
the colony-to-lawn transformation method. Then, the randomly selected transformants are checked for protein expression by SDS-PAGE. Plasmids
are extracted for positive clones, and DNA sequencing or next round of mutagenesis was performed (shown as dotted line). (b) The chemical
transformation method used for the same purpose. Plasmids are extracted from randomly selected clones after mutant library construction and
transformed into competent cells of expression strain for protein expression and SDS-PAGE analysis. The plasmids extracted from the clones which
have expected protein expression are used for DNA sequencing or next round of mutagenesis (shown as dotted line).
ª 2010 The Authors
Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 101
5. How to make transformation more efficient Y. An et al.
expected protein expression are used to extract plasmids incorrect protein expression in E. coli. Therefore, expres-
from their remaining cultures, and DNA sequencing or sion of proteins (especially for the well expressed
another round of mutagenesis was performed Although proteins) can be used to predict whether the genes are
the current transformation methods can be used for the in-frame, which can be further determined by DNA
same purpose, the process should be less convenient, sequencing. This strategy is reasonable because less
because more time and an additional experimental step plasmids need to be extracted for DNA sequencing.
(competent cell preparation) are needed (Fig. 2b). Addi- Therefore, a process based on colony-to-lawn transforma-
tionally, more plasmids should to be extracted, because tion and protein expression provides a convenient way to
the clones used for plasmid extraction are before protein screen in-frame clones from mutant libraries.
expression screening. In this work, the recombinant plas- In conclusion, as a simple and convenient DNA trans-
mids with random mutations of P450-BM3 gene intro- formation strategy, this method may find wide applica-
duced by error-prone PCR were used to test this method. tions in bioscience and biotechnology, especially when
The recombinant plasmids were changed hosts from clon- changing hosts of multiple plasmids is needed.
ing strain JM109 to expression strain BL21(DE3) with the
colony-to-lawn transformation method. Then, ten randomly
Acknowledgements
selected transformants were used to check protein expres-
sion levels, five of them were found to have expected The authors thank Sergi Castellano and Promdonkoy
protein expression. The plasmids were extracted and Patcharee for helpful discussions and review of this man-
DNA sequencing was performed, and as a result, all the uscript.
DNA sequences of positive clones were found to be in the
correct open reading frames.
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Discussion
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