transformation of plasmid dna into e. coli using the heat shock method

Transformación de ADN plásmido en E. coli usando el método de choque térmico es una técnica básica de la biología molecular. Consiste en la inserción de un producto extranjero plásmido o la ligadura en las bacterias. Este protocolo de vídeo describe el método tradicional de transformación utilizando comercialmente disponibles a partir de bacterias competentes químicamente Genlantis. Después de un corto de incubación en hielo, una mezcla de químicos y bacterias competentes de ADN se coloca a 42 ° C durante 45 segundos (golpe de calor) y luego se coloca de nuevo en hielo. SOC medios de comunicación y se añade a las células transformadas se incubaron a 37 º C durante 30 minutos con agitación. Para estar seguro de aislar a las colonias, independientemente de la eficiencia de transformación, dos cantidades de bacterias transformadas se siembran. Este protocolo tradicional puede ser utilizado con éxito para transformar bacterias competentes la mayoría de los disponibles en el mercado. El turbocells de Genlantis también puede ser utilizado en un protocolo de transformación la novela de 3 minutos, que se describe en el manual de instrucciones.

Watch this Scientific Journal Video about Transformation of Plasmid DNA into E. coli Using the Heat Shock Method at JoVE.com

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformación de ADN plásmido en E. coli utilizando el método de choque térmico

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

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Video: Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Expresión, aislamiento y purificación de soluble e insoluble Biotinylated Proteínas del Tejido Nervioso para regeneración

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Investigación

Bioingeniería

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagénesis dirigida casera de plásmidos enteros

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Biología

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Ingeniería 'Oro' de la fluorescencia por presión selectiva incorporación de aminoácidos no canónica y análisis de proteínas por espectrometría de masas y la fluorescencia

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

La identificación de un epítopo lineal de células B reconocido por anticuerpos monoclonales péptido Escaneo-asistida

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Inmunología e Infección

Evaluación de la actividad de las chaperonas Hsp70 utilizando células Escherichia coli dnaK756

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Autor destacado: Explorando las proteínas de choque térmico en las infecciones por malaria y tuberculosis

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

Ensayo de complementación basado en Escherichia coli para estudiar la función chaperona de la proteína de choque térmico 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

Bioquímica

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Protocolo rápido para la preparación de electrocompetentes<em&gt; Escherichia coli</em&gt; Y<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

Uso de ensamblaje in vivo para la construcción de plásmidos de alta eficiencia

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

Bacterial Transformation

<h4>Background</h4>

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the Streptococcus pneumoniae: a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule2. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died2. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a &#8220;transformation&#8221; process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation3.

Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith&#8217;s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes4.

<h4>Bacterial Transformation using Plasmids</h4>

Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it3,5. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA and generally provide certain functional benefits, such as resistance to antibiotics6,7. In their natural environment, bacteria undergo &#8220;bacterial transformation&#8221; by taking in plasmids from other bacteria in a process called conjugation8. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.

Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create &#8220;recombinant plasmids&#8221; that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane. One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution9. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.

<h4>Applications</h4>

Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools10. In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids11. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids12. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients10. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation10. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers13. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.

<h4>References</h4>

<ol class="references">
	<li>GL, Armstrong, LA, Conn and RW, Pinner. Trends in Infectious Disease Mortality in the United States During the 20th Century. JAMA. 1999, Vol. 281, 1 (61-66).</li>
	<li>F, Griffith. The Significance of Pneumococcal Types. J Hyg (Lond). . 1928 , 2 (113-59).</li>
	<li>Lorenz, MG and Wackernagel, F. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev. . 1994 Sep;, Vol. 58, 3: (563-602).</li>
	<li>S, Domingues, et al. Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. PLOS Pathogens. 2012, 8(8): e1002837.</li>
	<li>Dubnau, D. DNA uptake in bacteria. Annu Rev Microbiol. . 1999, 53: (217-44).</li>
	<li>Solar, G del, et al. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. . 1998 , Vol. 62, 2: (434-64).</li>
	<li>Bennett, PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. . 2008 , Vol. 153, S1: (S347-S357).</li>
	<li>MLlosa, et al. Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol. 2002 Jul;, Vol. 45, 1: (1-8).</li>
	<li>Roychoudhury, A, Basu, S and Sengupta, DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian J Biochem Biophys. . 2009 , Vol. 46, 5: (395-400).</li>
	<li>Khan, S, et al. Role of Recombinant DNA Technology to Improve Life. I. nt J Genomics. . 2016, 2016:2405954.</li>
	<li>Marsischky, G and LaBaer, J. Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. Genome Res. 2004, Vol. 14, 2020-28.</li>
	<li>Doudna, JA and Charpentie, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014, Vol. 346, 6213-1258096.</li>
	<li>Good, A. Toward nitrogen-fixing plants. Science. 2018, Vol. 359, 6378: 869-70.</li>
</ol>

Bacterial Transformation using Plasmids - Concept

Transformación bacteriana

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

Transformación de ADN plásmido en E. coli utilizando el método de choque térmico

Resumen

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Ensayo de complementación basado en Escherichia coli para estudiar la función chaperona de la proteína de choque térmico 70

Ensayo de complementación basado en Escherichia coli para estudiar la función chaperona de la proteína de choque térmico 70

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Protocolo rápido para la preparación de electrocompetentes

Uso de ensamblaje in vivo para la construcción de plásmidos de alta eficiencia

Transformación bacteriana