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
Proteomics studies on Arabidopsis ThalianaGaurav Dwivedi
This document describes a study analyzing cell wall proteins during xylem vessel secondary cell wall formation in cell culture. The objective was to identify proteins involved in secondary cell wall growth and lignification. Researchers used cell cultures of Arabidopsis thaliana treated with hormones to induce tracheary element differentiation. They extracted proteins from cell walls using various salt concentrations and identified proteins using mass spectrometry. The results provided insights into how induction hormones may affect association of cell wall proteins during secondary cell wall formation.
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.
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.
Karen Hatten Experiment II Final ReportKaren Hatten
This experiment transformed the cyanobacterium Synechocystis sp. PCC 6803 in two parts. Part A introduced a mutation to the psbC gene, which encodes a chlorophyll-binding protein, via a plasmid. This disrupted photosystem II and allowed selection of transformed cells. Part B amplified the wild-type psbC gene, cloned it into a plasmid, and transformed mutant cells to restore photosystem function. Various DNA manipulations, transformations, and selections were performed to characterize and select transformed cells at each step.
Through genetic barcoding of the tufA gene, an unknown green algae specimen was identified as belonging to the Ulva compressa clade. DNA was isolated from the specimen and the tufA gene was amplified via PCR. The gene was cloned and the nucleotide sequence analyzed and compared to sequences in GenBank. Phylogenetic analysis indicated the specimen was most similar to Ulva sp. BER-2007, but could not be identified to the species level. While in the U. compressa clade, further testing is needed to confirm its identity as a potential new species of Ulva in Narragansett Bay.
1. Researchers developed a new Disk Carbapenemase Test (DCT) using paper disks containing imipenem and a pH indicator to rapidly detect carbapenemase-producing gram-negative bacilli (GNB).
2. The DCT was evaluated on 742 GNB strains and detected 98.6% of strains with KPC, NDM, IMP, VIM, and SIM carbapenemases within 60 minutes. However, it did not detect some strains with OXA and GES carbapenemases.
3. The DCT showed 100% specificity in tests on 448 carbapenem-resistant GNB that were not tested for carbapenemase genes. The DCT provides a simple, rapid method for detecting important
This document describes a study on expressing recombinant nanobodies in E. coli cells, extracting the proteins, and purifying them. E. coli WK6 cells were transformed with a plasmid containing the nanobody gene and expression was induced with IPTG. The cells were lysed and the proteins in the periplasmic space were extracted. Purification was done using immobilized metal affinity chromatography to bind the histidine-tagged nanobody, which was then eluted with imidazole buffer. SDS-PAGE and Western blot were used to analyze the expression and purity of the nanobody.
1) Phenolic disinfectants like phenol, 2,4-dichlorophenol, and p-tert-amylphenol bound to Micrococcus lysodeikticus cells, with higher percentages binding to cells for more potent disinfectants.
2) Protoplasts bound slightly less (around 20%) of the phenolic disinfectants compared to whole cells, suggesting cell walls contribute to binding.
3) Binding of 2,4-dichlorophenol decreased with increasing pH, while binding of phenol and p-tert-amylphenol was constant over the pH range tested, relating to differences in ionization properties.
Proteomics studies on Arabidopsis ThalianaGaurav Dwivedi
This document describes a study analyzing cell wall proteins during xylem vessel secondary cell wall formation in cell culture. The objective was to identify proteins involved in secondary cell wall growth and lignification. Researchers used cell cultures of Arabidopsis thaliana treated with hormones to induce tracheary element differentiation. They extracted proteins from cell walls using various salt concentrations and identified proteins using mass spectrometry. The results provided insights into how induction hormones may affect association of cell wall proteins during secondary cell wall formation.
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.
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.
Karen Hatten Experiment II Final ReportKaren Hatten
This experiment transformed the cyanobacterium Synechocystis sp. PCC 6803 in two parts. Part A introduced a mutation to the psbC gene, which encodes a chlorophyll-binding protein, via a plasmid. This disrupted photosystem II and allowed selection of transformed cells. Part B amplified the wild-type psbC gene, cloned it into a plasmid, and transformed mutant cells to restore photosystem function. Various DNA manipulations, transformations, and selections were performed to characterize and select transformed cells at each step.
Through genetic barcoding of the tufA gene, an unknown green algae specimen was identified as belonging to the Ulva compressa clade. DNA was isolated from the specimen and the tufA gene was amplified via PCR. The gene was cloned and the nucleotide sequence analyzed and compared to sequences in GenBank. Phylogenetic analysis indicated the specimen was most similar to Ulva sp. BER-2007, but could not be identified to the species level. While in the U. compressa clade, further testing is needed to confirm its identity as a potential new species of Ulva in Narragansett Bay.
1. Researchers developed a new Disk Carbapenemase Test (DCT) using paper disks containing imipenem and a pH indicator to rapidly detect carbapenemase-producing gram-negative bacilli (GNB).
2. The DCT was evaluated on 742 GNB strains and detected 98.6% of strains with KPC, NDM, IMP, VIM, and SIM carbapenemases within 60 minutes. However, it did not detect some strains with OXA and GES carbapenemases.
3. The DCT showed 100% specificity in tests on 448 carbapenem-resistant GNB that were not tested for carbapenemase genes. The DCT provides a simple, rapid method for detecting important
This document describes a study on expressing recombinant nanobodies in E. coli cells, extracting the proteins, and purifying them. E. coli WK6 cells were transformed with a plasmid containing the nanobody gene and expression was induced with IPTG. The cells were lysed and the proteins in the periplasmic space were extracted. Purification was done using immobilized metal affinity chromatography to bind the histidine-tagged nanobody, which was then eluted with imidazole buffer. SDS-PAGE and Western blot were used to analyze the expression and purity of the nanobody.
1) Phenolic disinfectants like phenol, 2,4-dichlorophenol, and p-tert-amylphenol bound to Micrococcus lysodeikticus cells, with higher percentages binding to cells for more potent disinfectants.
2) Protoplasts bound slightly less (around 20%) of the phenolic disinfectants compared to whole cells, suggesting cell walls contribute to binding.
3) Binding of 2,4-dichlorophenol decreased with increasing pH, while binding of phenol and p-tert-amylphenol was constant over the pH range tested, relating to differences in ionization properties.
The document summarizes a student laboratory experiment attempting to genetically transform E. coli bacterial cells with a GFP plasmid (pGLO) using heat shock. The expected results were that bacterial cells transformed with the plasmid would grow on ampicillin-containing media and glow under black light. However, the actual results found that all bacterial cells died on ampicillin-containing plates, contradicting expectations. Possible sources of error that could have caused transformation failure are discussed.
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
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 summarizes a lab experiment comparing participants' genotypes and phenotypes for tasting the bitter compound phenylthiocarbamide (PTC). The results showed that the genotype determined by PCR and gel electrophoresis matched the self-reported PTC tasting phenotype for all participants. Specifically, those with the tt genotype could not taste PTC, while those with the TT or Tt genotypes reported PTC as bitter tasting. The lab demonstrated that an individual's ability to taste PTC is inherited in a dominant/recessive manner governed by the TAS2R38 gene.
The research article published in the journal RSC Advances in 2017, entitled as "One step preparation of a biocompatible,
antimicrobial reduced graphene oxide–silver nanohybrid as a topical antimicrobial agent"
BLIRT is a biotechnology company in Gdansk, Poland that offers outsourced R&D services including biology and chemistry services. It has modern laboratories and a team of over 30 employees, including 22 researchers of which 9 have PhDs. BLIRT has the equipment and expertise to handle projects of all sizes, from gene synthesis to protein production and purification to analytical testing. It works with clients through different collaboration models including fee-for-service, co-development, FTE, and consulting. Key benefits include competitive pricing, short deadlines, scientific expertise, and state-of-the-art facilities.
This document is a thesis submitted by Sunehera Sarwat to the University of Nottingham for a Masters in Research degree. It discusses trials and tribulations in expressing and purifying the ABCG2 transporter protein. The author initially tried expressing ABCG2 tagged with His6 in insect cells, but was unable to purify it using nickel resins, likely due to occlusion of the tag. Alternative constructs with Strep and Strep-His13 tags were made, but these also could not be purified. Detergent solubilization allowed for partial purification of Strep-His13 tagged ABCG2. The thesis aims to develop robust methods for purifying ABCG2 to further structural studies of this important multidrug
The document summarizes several biology lab experiments conducted by the author:
1. They performed DNA extraction from samples, PCR, and Western blot techniques. For DNA extraction, their sample did not produce the expected results.
2. They learned aseptic technique and used Gram staining to identify bacteria samples as gram positive or negative.
3. PCR was used to amplify genomic DNA between primers over multiple cycles. Controls were included.
4. Nested PCR with more specific primers was used to further amplify portions of DNA. Exonuclease treated samples before nested PCR.
5. Gel electrophoresis separated DNA fragments by size. PCR products from two plant samples were analyzed, with one showing bands.
1. Serratia marcescens was found to produce prodigiosin when cultured in nutrient broth, with an estimated production of 1516.9 prodigiosin units per cell. 2. Characterization of the isolated red pigment indicated it was prodigiosin through analyses including TLC, HPLC, and biochemical tests. 3. Prodigiosin demonstrated antimicrobial activity against test pathogens and cytotoxic effects on cancer cell lines in a dose-dependent manner, suggesting specific anti-neoplastic activity.
The document summarizes a workshop held on February 11, 2021 at Smt. Kasturbai Walchand College in Sangli to demonstrate experiments from the revised syllabus of the B.Sc. III practical course. It includes demonstrations and explanations of experiments on isolating plant genomic DNA and estimating the DNA using diphenylamine. It also discusses the steps involved in genetically engineering golden rice through the introduction of genes from daffodil and bacteria to allow the rice plant to produce beta-carotene.
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.
Abstract
Objective(s):
The development of reliable and ecofriendly process for the synthesis of nano-metals is an important aspect in the field of nanotechnology. Nano-metals are a special group of materials with broad area of applications.
Materials and Methods:
In this study, extracellular synthesis of silver nanoparticles (SNPs) performed by use of the gram positive soil Streptomycetes. Streptomycetes isolated from rice fields of Guilan Province, Iran (5 isolates). Initial characterization of SNPs was performed by visual change color. To determine the bacterium taxonomical identity, its colonies characterized morphologically by use of scanning electron microscope. The PCR molecular analysis of active isolate represented its identity partially. In this regard, 16S rDNA of isolate G was amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then conducted using NCBI GenBank database using BLAST. Also SNPs were characterized by, transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD).
Results:
From all 5 collected Streptomyces somaliensis isolates, isolate G showed highest extracellular synthesis of SNPs via in vitro. SNPs were formed immediately by the addition of (AgNO3) solution (1 mM). UV-visible spectrophotometry for measuring surface plasmon resonance showed a single absorption peak at 450 nm, which confirmed the presence of SNPs. TEM revealed the extracellular formation of spherical silver nanoparticles in the size range of 5-35 nm.
Conclusions:
The biological approach for the synthesis of metal nanoparticles offers an environmentally benign alternative to the traditional chemical and physical synthesis methods. So, a simple, environmentally friendly and cost-effective method has been developed to synthesize AgNPs using Streptomycetes.
1. The study isolated the pigment prodigiosin from the soil bacterium Serratia marcescens and characterized it through techniques like TLC and HPLC.
2. Prodigiosin was tested for its antimicrobial activity against bacterial strains and exhibited inhibition of their growth. It also showed dose-dependent cytotoxic effects on cancer cell lines but not normal cell lines, indicating specific anti-cancer activity.
3. The study demonstrated prodigiosin's ability to stain DNA on agarose gel electrophoresis from concentrations as low as 3 μg, with effective staining between 10-40 μg and DNA fragmentation at 50 μg.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
1. The document describes a study that isolated Bacillus species bacteria from malathion contaminated soil that is capable of degrading malathion.
2. The isolated bacteria were identified through morphological, cultural, and biochemical analysis as belonging to the Bacillus species.
3. When the isolated Bacillus species bacteria were added to soil supplemented with malathion and incubated, the phosphate levels increased, indicating the bacteria's ability to degrade malathion.
This document describes experiments to produce and characterize the D4 protein binding domain of Clostridium botulinum toxin. The D4 protein was expressed in E. coli and purified using glutathione resin. It was then incubated with N2A cells that had been transfected with baculovirus to label early endosomes. Confocal microscopy showed that Alexa Fluor-labeled D4 protein bound to the endosomes of the N2A cells in a concentration-dependent manner.
The document discusses bacterial keratinases, which are enzymes produced by certain bacteria, actinomycetes and fungi. These enzymes are useful for bioprocessing agricultural and industrial wastes containing keratin, such as poultry feathers. Keratinases can degrade keratin proteins through the cleavage of disulfide bonds and are used for the conversion of poultry feathers into feather meal. Some keratinases are serine proteases while others are metalloproteases. These enzymes show potential for more sustainable processing of keratin wastes and animal hides in leather production compared to conventional chemical methods.
100520 fluidization past and future, plenary by horio at fluidization xiiiMasayuki Horio
The lecture consists of two parts:
1. Introduction of my recent activity at JST-RISTEX on community based activities against global warming
2. Historical perspective of fluidization science and engineering
In the latter a unique discussion was attempted on the structure of nature (existing things) and the 3 stage law in paradigm shift in scientific research. The history of fluidization research was then analysed in terms of the three stage law.
This document presents information about fluidization and was prepared by 5 students. It defines fluidization as a process where solids are made to behave like fluids by passing gas or liquid upwards. Fluidization is widely used in industries for operations like transportation, heating, mixing and chemical reactions using catalysts. The document discusses fluidization regimes from fixed beds to entrained beds as gas velocity increases. It also describes Geldart's classification of powders and common applications of fluidization like fluid catalytic cracking in petroleum industry.
Fluidized bed introduction by mohabat ali malik(MUET,jamshoro)mohabat_ali
This document provides an introduction to fluidized beds, including their components, advantages, disadvantages, applications, and flow regimes. It describes how fluidized beds are composed of a solid material (typically a catalyst) that is fluidized by a gas or liquid. The document outlines the main flow regimes - bubbling, turbulent, and fast fluidization - and discusses factors that influence transitions between regimes like pressure, temperature, and particle properties. It also provides examples of industrial processes using fluidized beds and references for further reading.
The document summarizes a student laboratory experiment attempting to genetically transform E. coli bacterial cells with a GFP plasmid (pGLO) using heat shock. The expected results were that bacterial cells transformed with the plasmid would grow on ampicillin-containing media and glow under black light. However, the actual results found that all bacterial cells died on ampicillin-containing plates, contradicting expectations. Possible sources of error that could have caused transformation failure are discussed.
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
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 summarizes a lab experiment comparing participants' genotypes and phenotypes for tasting the bitter compound phenylthiocarbamide (PTC). The results showed that the genotype determined by PCR and gel electrophoresis matched the self-reported PTC tasting phenotype for all participants. Specifically, those with the tt genotype could not taste PTC, while those with the TT or Tt genotypes reported PTC as bitter tasting. The lab demonstrated that an individual's ability to taste PTC is inherited in a dominant/recessive manner governed by the TAS2R38 gene.
The research article published in the journal RSC Advances in 2017, entitled as "One step preparation of a biocompatible,
antimicrobial reduced graphene oxide–silver nanohybrid as a topical antimicrobial agent"
BLIRT is a biotechnology company in Gdansk, Poland that offers outsourced R&D services including biology and chemistry services. It has modern laboratories and a team of over 30 employees, including 22 researchers of which 9 have PhDs. BLIRT has the equipment and expertise to handle projects of all sizes, from gene synthesis to protein production and purification to analytical testing. It works with clients through different collaboration models including fee-for-service, co-development, FTE, and consulting. Key benefits include competitive pricing, short deadlines, scientific expertise, and state-of-the-art facilities.
This document is a thesis submitted by Sunehera Sarwat to the University of Nottingham for a Masters in Research degree. It discusses trials and tribulations in expressing and purifying the ABCG2 transporter protein. The author initially tried expressing ABCG2 tagged with His6 in insect cells, but was unable to purify it using nickel resins, likely due to occlusion of the tag. Alternative constructs with Strep and Strep-His13 tags were made, but these also could not be purified. Detergent solubilization allowed for partial purification of Strep-His13 tagged ABCG2. The thesis aims to develop robust methods for purifying ABCG2 to further structural studies of this important multidrug
The document summarizes several biology lab experiments conducted by the author:
1. They performed DNA extraction from samples, PCR, and Western blot techniques. For DNA extraction, their sample did not produce the expected results.
2. They learned aseptic technique and used Gram staining to identify bacteria samples as gram positive or negative.
3. PCR was used to amplify genomic DNA between primers over multiple cycles. Controls were included.
4. Nested PCR with more specific primers was used to further amplify portions of DNA. Exonuclease treated samples before nested PCR.
5. Gel electrophoresis separated DNA fragments by size. PCR products from two plant samples were analyzed, with one showing bands.
1. Serratia marcescens was found to produce prodigiosin when cultured in nutrient broth, with an estimated production of 1516.9 prodigiosin units per cell. 2. Characterization of the isolated red pigment indicated it was prodigiosin through analyses including TLC, HPLC, and biochemical tests. 3. Prodigiosin demonstrated antimicrobial activity against test pathogens and cytotoxic effects on cancer cell lines in a dose-dependent manner, suggesting specific anti-neoplastic activity.
The document summarizes a workshop held on February 11, 2021 at Smt. Kasturbai Walchand College in Sangli to demonstrate experiments from the revised syllabus of the B.Sc. III practical course. It includes demonstrations and explanations of experiments on isolating plant genomic DNA and estimating the DNA using diphenylamine. It also discusses the steps involved in genetically engineering golden rice through the introduction of genes from daffodil and bacteria to allow the rice plant to produce beta-carotene.
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.
Abstract
Objective(s):
The development of reliable and ecofriendly process for the synthesis of nano-metals is an important aspect in the field of nanotechnology. Nano-metals are a special group of materials with broad area of applications.
Materials and Methods:
In this study, extracellular synthesis of silver nanoparticles (SNPs) performed by use of the gram positive soil Streptomycetes. Streptomycetes isolated from rice fields of Guilan Province, Iran (5 isolates). Initial characterization of SNPs was performed by visual change color. To determine the bacterium taxonomical identity, its colonies characterized morphologically by use of scanning electron microscope. The PCR molecular analysis of active isolate represented its identity partially. In this regard, 16S rDNA of isolate G was amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then conducted using NCBI GenBank database using BLAST. Also SNPs were characterized by, transmission electron microscopy (TEM) and X-ray diffraction spectroscopy (XRD).
Results:
From all 5 collected Streptomyces somaliensis isolates, isolate G showed highest extracellular synthesis of SNPs via in vitro. SNPs were formed immediately by the addition of (AgNO3) solution (1 mM). UV-visible spectrophotometry for measuring surface plasmon resonance showed a single absorption peak at 450 nm, which confirmed the presence of SNPs. TEM revealed the extracellular formation of spherical silver nanoparticles in the size range of 5-35 nm.
Conclusions:
The biological approach for the synthesis of metal nanoparticles offers an environmentally benign alternative to the traditional chemical and physical synthesis methods. So, a simple, environmentally friendly and cost-effective method has been developed to synthesize AgNPs using Streptomycetes.
1. The study isolated the pigment prodigiosin from the soil bacterium Serratia marcescens and characterized it through techniques like TLC and HPLC.
2. Prodigiosin was tested for its antimicrobial activity against bacterial strains and exhibited inhibition of their growth. It also showed dose-dependent cytotoxic effects on cancer cell lines but not normal cell lines, indicating specific anti-cancer activity.
3. The study demonstrated prodigiosin's ability to stain DNA on agarose gel electrophoresis from concentrations as low as 3 μg, with effective staining between 10-40 μg and DNA fragmentation at 50 μg.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
1. The document describes a study that isolated Bacillus species bacteria from malathion contaminated soil that is capable of degrading malathion.
2. The isolated bacteria were identified through morphological, cultural, and biochemical analysis as belonging to the Bacillus species.
3. When the isolated Bacillus species bacteria were added to soil supplemented with malathion and incubated, the phosphate levels increased, indicating the bacteria's ability to degrade malathion.
This document describes experiments to produce and characterize the D4 protein binding domain of Clostridium botulinum toxin. The D4 protein was expressed in E. coli and purified using glutathione resin. It was then incubated with N2A cells that had been transfected with baculovirus to label early endosomes. Confocal microscopy showed that Alexa Fluor-labeled D4 protein bound to the endosomes of the N2A cells in a concentration-dependent manner.
The document discusses bacterial keratinases, which are enzymes produced by certain bacteria, actinomycetes and fungi. These enzymes are useful for bioprocessing agricultural and industrial wastes containing keratin, such as poultry feathers. Keratinases can degrade keratin proteins through the cleavage of disulfide bonds and are used for the conversion of poultry feathers into feather meal. Some keratinases are serine proteases while others are metalloproteases. These enzymes show potential for more sustainable processing of keratin wastes and animal hides in leather production compared to conventional chemical methods.
100520 fluidization past and future, plenary by horio at fluidization xiiiMasayuki Horio
The lecture consists of two parts:
1. Introduction of my recent activity at JST-RISTEX on community based activities against global warming
2. Historical perspective of fluidization science and engineering
In the latter a unique discussion was attempted on the structure of nature (existing things) and the 3 stage law in paradigm shift in scientific research. The history of fluidization research was then analysed in terms of the three stage law.
This document presents information about fluidization and was prepared by 5 students. It defines fluidization as a process where solids are made to behave like fluids by passing gas or liquid upwards. Fluidization is widely used in industries for operations like transportation, heating, mixing and chemical reactions using catalysts. The document discusses fluidization regimes from fixed beds to entrained beds as gas velocity increases. It also describes Geldart's classification of powders and common applications of fluidization like fluid catalytic cracking in petroleum industry.
Fluidized bed introduction by mohabat ali malik(MUET,jamshoro)mohabat_ali
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Chemical and Physical properties of Cassava Starch-Cm-Chitosan-Acrylic Acid Hydrogel prepared from radiation –induced crosslinking
Gatot Trimulyadi Rekso
Center for Application of Isotopes and Radiation- National Nuclear Energy Agency
Jl. Lebak Bulus Raya No. 49, Jakarta-Selatan, Indonesia
Corresponding author; e-mail; gatot2811@yahoo.com ,
Fax: +62-21-.7513270, HP ; 08129419442
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Abstract
Objective(s):
Although viral vectors are considered efficient gene transfer agents, their board application has been limited by toxicity, immunogenicity, mutagenicity and small gene carrying capacity. Non-viral vectors are safe but they suffer from low transfection efficiency. In the present study, polyallylamine (PAA) in two molecular weights (15 and 65 kDa) was modified by alkane derivatives in order to increase transfection activity and to decrease cytotoxicity.
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Glass bead transformation method for gram positive bacteria
1. Brazilian Journal of Microbiology (2009) 40: 923-926
ISSN 1517-8382
GLASS BEAD TRANSFORMATION METHOD FOR GRAM-POSITIVE BACTERIA
Pongsak Rattanachaikunsopon*, Parichat Phumkhachorn
Department of Biological Science, Ubon Ratchathani University, Warin Chamrap, Ubon Ratchathani 34190, Thailand
Submitted: February 10, 2009; Returned to authors for corrections: April 26, 2009; Approved: May 15, 2009.
ABSTRACT
A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria.
It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and
polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of
Gram-positive bacteria was achieved.
Key words: transformation; Gram-positive bacteria; glass bead
INTRODUCTION membranes. Under appropriate conditions, DNA present in
surrounding environment may enter through the pores. This
The difficulty in introducing DNA into cells of Gram- method was successfully used to introduce DNA into several
positive bacteria is one of the major factors that hinders strains of Gram-positive bacteria (1-4, 7-9, 11, 15, 16).
genetic studies of Gram-positive bacteria. The thick Although electroporation can be used to transform the
peptidoglycan layer in their cell walls is considered a bacteria with high efficiency, it cannot be performed by
potential barrier to DNA uptake. Transformation of these small and less-equipped laboratories because of the
organisms cannot occur naturally but can be accomplished requirement of expensive and specialized equipment. In this
by particular transformation methods such as protoplast study, glass bead transformation was proposed as a simple,
transformation and electroporation. Protoplast transformation inexpensive and reproducible transformation method for
was developed for enzymatic removal of the cell wall to Gram-positive bacteria.
create protoplasts. In the presence of polyethylene glycol, Glass bead transformation was performed by using of
DNA uptake by protoplasts is facilitated. It was developed Gram-positive bacteria as recipients for pGK12, a 4.4 kb E.
for some Gram-positive bacteria (5, 6, 10, 12, 14, 17). coli/Lactococcus shuttle vector carrying an erythromycin
Because of its low and highly variable efficiency and time resistance gene. The bacteria used in transformation
consuming protocol, this method is rarely used. Presently, experiments were Enterococcus faecalis TISTR 927,
electroporation is the most commonly used transformation Lactobacillus casei ATCC 393, Lactococcus lactis DSM
method for Gram-positive bacteria. It involves the 20481, Leuconostoc dextranicum ATCC 19255, Listeria
application of high voltage electric pulse of short duration to innocua DSM 20649, Staphylococcus aureus ATCC 25923
induce the formation of transient pores in cell walls and and Streptococcus pneumoniae ATCC 10015.
*Corresponding Author. Mailing address: Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Warin Chamrap, Ubon
Ratchathani 34190, Thailand.; E-mail: rattanachaikunsopon@yahoo.com
923
2. Rattanachaikunsopon, P. et al.
To obtain protoplasts of Gram-positive bacteria, cultures for Gram-positive bacteria with acceptable efficiency and
of the bacteria were grown at 37°C in 30 ml of BHI broth to high reproducibility.
mid log phase. Cells were harvested by centrifugation at
5,000 xg for 5 min, washed in distilled water, and suspended
Table 1. Numbers of transformants and transformation
in 5.0 ml of 0.5 M sorbitol in 0.01 M Tris-hydrochloride (pH
frequencies obtained from glass bead transformation of
7.0). The cell suspension was mixed with mutanolysin (at the
Gram-positive bacteria with pGK12
final concentration of 50 µg/ml) and then incubated at 37°C
for 30 min. Protoplasts were pelleted at 5,000 xg for 5 min, Gram-positive Number of Transformation
bacteria transformanats frequency
washed in 10 ml of transformation buffer (0.5 M sorbitol, (x 103)a (per µg of pGK12)
0.02 M maleate, 0.02 M MgCl2, pH 6.5), and resuspended in E. faecalis 3.8, 5.0, 4.4, 4.6, 5.2 4.60 x 103
1 ml of transformation buffer. L. casei 4.3, 4.0, 4.8, 5.0, 4.6 4.54 x 103
Glass bead transformation of Gram positive bacteria was L. lactis 6.9, 6.3, 7.1, 6.7, 6.1 6.62 x 103
conducted as follows. Aliquots of 0.5 ml of protoplast L. dextranicum 5.8, 6.1, 4.8, 5.7, 5.0 5.48 x 103
suspension were placed in 15 ml conical disposable L. innocua 5.1, 4.7, 4.5, 5.3, 4.0 4.72 x 103
polypropylene centrifuge tubes (Corning, USA). One µg of S. aureus 3.3, 3.8, 4.2, 3.0, 3.5 3.56 x 103
pGK12 was added to the protoplasts, followed immediately S. pneumoniae 3.6, 4.8, 4.3, 3.2, 4.5 4.08 x 103
a
by the addition of 500 µl of 30% polyethylene glycol 6,000 obtained from 5 separate experiments
(PEG 6,000) and 0.3 g of acid washed glass beads (212-300
µm in diameter, Sigma, USA). Protoplasts were agitated at Some parameters were tested for their effects on
the highest speed on a vortex mixer for 15 sec, and then transformation including amount of plasmid DNA,
diluted by the addition of 10 ml of transformation buffer. concentration and molecular weight of PEG and agitation
After the beads were allowed to settle, agitated protoplast time. Since L. lactis gave the highest transformation
suspension was transferred to a new 15 ml conical tube. Cells frequency in glass bead transformation, it was used as a
were pelleted by centrifugation at 5,000 xg for 5 min and recipient for pGK12 in the following experiments.
suspended in 1 ml of BHI supplemented with 0.5 M sorbitol. Quantities of pGK12 DNA ranging from 0.5 to 5.0 µg
After incubation at 37°C for 1 h, transformants were were used in glass bead transformation of L. lactis. Results
recovered by plating the protoplast culture onto BHI agar indicated that transformation frequencies were somewhat
containing 0.5 M sorbitol and erythromycin at the final stable at the highest level when DNA between 1.0 and 3.0 µg
concentration of 3 µg/ml and incubated at 37°C for 5 to 7 was used. Interestingly, increase of the amount of pGK12
days. Transformation frequency was expressed as above 3.0 µg caused higher numbers of transformants
erythromycin resistant colonies per 1 µg of pGK12, and recovered but lower numbers of transformation frequency
values reported represented the mean from 5 separate (Fig. 1). It is not known if this is due to an inappropriate
experiments. Transformation frequencies and numbers of proportion of amount of protoplasts to amount of DNA used
transformants obtained from these experiments are shown in in the transformation. Similar result was found in protoplast
Table 1. The presence of pGK12 in transformants was transformation of Enterococcus faecalis OG1RF with
confirmed by Southern hybridization analysis using DIG- pGB354. It was reported that increase of plasmid
labeled pGK12 as a probe. These results suggest that glass concentration beyond 0.5 µg per ml of transformation
bead transformation can be used as transformation method mixture caused reduction of transformation frequency (17).
924
3. Glass bead transformation method
recovered in the glass bead transformation without agitation
70 250
or with agitation for 30 sec (Fig. 2). Protoplasts of L. lactis
No. of transformant (x100 CFU)
60
Transformation frequency (x100)
were also mixed with 15% PEG and 1 µg of pGK12 without
200
50 any agitation for 10, 20, 30, 40, 50 and 60 min. No
40
150 transformant was observed in every mixing time. The results
suggest that the transformation cannot occur naturally and
30
100
the agitation of protoplasts with glass beads may be
20
necessary for the process to occur.
50
10
0 0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 70
Transformation frequency (x100)
A ount of plasm (µg)
m id
60
50
Figure 1. Transformation frequencies and numbers of 40
transformants obtained from glass bead transformation of L.
30
lactis with different amounts of pGK12.
20
10
Glass bead transformation of L. lacis were designed to
0
use PEG with 3 different molecular weights (3,350, 6,000 0 5 10 15 20 25 30
and 8,000) at various concentrations including 0%, 5%, 10%,
Agitation tim (sec)
e
15%, 20%, 25% and 30%. Molecular weight of PEG was
shown not to have any effect on transformation. However, Figure 2. Transformation frequencies obtained from glass
transformation frequency was found to be affected by bead transformation of L. lactis with pGK12 using different
concentration of PEG. The highest transformation frequency agitation times.
was observed in the transformation using 10% PEG. The
optimal concentrations of PEG for protoplast transformation
of Streptomyces spp., Streptococcus lactis, Bacillus subtilis In sum, we would like to stress that this is the first report
and Enterococcus faecalis OG1X were reported to be 20% of the transformation of Gram-positive bacteria using glass
(2), 22.5% (6), 30% (2) and 40% (17), respectively. bead. Because of its convenience, cost saving, high
Agitation protoplasts with glass beads may be the reason for reproducibility and acceptable efficiency, it may be an
the reduction of concentration of PEG used in glass bead alternative method for transformation of Gram-positive
transformation. bacteria with plasmid DNA.
Effect of agitation time on transformation was evaluated.
Protoplasts of L. lacis subjected to glass bead transformation ACKNOWLEDGEMENTS
were agitated with glass beads for different periods of time
ranging from 0 to 30 sec. Transformation was most efficient We are grateful to Dr. Jan Kok, University of
between 10 and 15 sec of agitation. No transformants was Groningen, the Netherlands for providing pGK12.
925
4. Rattanachaikunsopon, P. et al.
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