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Heat shock vs electroporation

Heat shock vs electroporation


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I've been transforming E. coli via heat shock in order to insert oligonucleotides (around 50 nt); however, none of my experiments have given positive results so far. I begin to question the efficiency of chemical transformation, especially for short DNA fragments. Is there such a notable difference between chemical and electro transformation?

EDIT: I'm checking for CRISPR array expansion, so my oligos have a protospacer length and structure. I'm using BL21(DE3) cells which have an IPTG-inducible T7 that I'm first transforming with a Cas1-Cas2 plasmid. Then, I induce the cells in order to express those proteins, make them competent, and transform then once again but with the oligos and an eGFP plasmid (for some sort of selection). To check for positive results, I'm amplifying the leader-repeat junction of the CRISPR array and compare its length (via electrophoresis) with a usual array that has no integrands.


How do you detect a potenially positive transfection? Short fragments might have proper teriary structures which may make them behave differently than you would expect. Also, noncircular DNA and RNA is targeted much faster for degradation in most cells if the right head or tail signals are missing.

To your question: pulsed electroporation with well prepared bacteria (state, buffer,… ) is usually 5-100 x more efficient than chemically competent bacteria.


Dilute the ligation reaction for electroporation - (Jun/18/2008 )

I do so many time the electroporation for BL21 E.coli by 1ul direct ligation but in electroporator always flash come so is it mportant to dilute the ligation reaction and what the best way to dilute it.

Yep, dilute it with ddH20 10 times, and use 1ul.

Electroporation is very efficient method (Vs. heat shock), so using diluted ligation reaction will not be a problem. In fact, in my experience, it incereses the efficiency.

Yep, dilute it with ddH20 10 times, and use 1ul.

Electroporation is very efficient method (Vs. heat shock), so using diluted ligation reaction will not be a problem. In fact, in my experience, it incereses the efficiency.

thank u for ur idea today i did transformation with diluted mixture and no flash and tommorow I will check the no. of colony thank u

Yep, dilute it with ddH20 10 times, and use 1ul.

Electroporation is very efficient method (Vs. heat shock), so using diluted ligation reaction will not be a problem. In fact, in my experience, it incereses the efficiency.

thank u for ur idea today i did transformation with diluted mixture and no flash and tommorow I will check the no. of colony thank u

daer cellcounter
am not happay because even in control plate no colony what can I do

A suggestion. Float small milipore filter paper on dd-water in a petri-dish..and simply put 5-10uL of your ligation mixture on them for an hour or so. After that recover the ligation mix with a pipette. and go for transformation. Principle as you might have guessed by now is "micro" dialysis for desalting..hopefully it works. do let me know..

1. There are several other things that may be going wrong, so, you need to troubleshoot other aspects too. Starting from the bacterial strain, media, antibiotics, electroporator, cuvettes, incubation times etc etc. As you are already doing control plasmid electroporation, you may be fine without troubleshooting cloning part (ligation etc), but make sure the controls you are doing cover every eventuality.

It is a very bad idea to try to transform BL21 strains directly with a ligation product. You will save time and grief by transforming first into DH5a, DH10B or another cloning strain, doing a miniprep, and transforming the BL21 with the plasmid DNA. BL21 has very poor transformation efficiency.


Biological effects of electric shock and heat denaturation and oxidation of molecules, membranes, and cellular functions

Direct exposure of cells in suspension to intense electric pulses is known to produce damages to cell membranes and supramolecular organizations of cells, and denaturation of macromolecules, much like injuries and tears seen in electric trauma patients. Thus, the system has been used as a laboratory model for investigating the biochemical basis of electric injury. An intense electric pulse can produce two major effects on cells--one caused by the field, or the electric potential, and the other by current, or the electric energy. The field-induced transmembrane potential can produce electro-conformational changes of ion channels and ion pumps and, when the potential exceeds the dielectric strength of the cell membrane (approximately 500 mV for a pulse width of a few ms), electro-conformational damages and electroporations of membrane proteins and lipid bilayers. These events lead to passage of electric current through the membrane-porated cells and to heating of cell membranes and cytoplasmic contents. The subsequent denaturation of cell membranes and cytoplasmic macromolecules brings about many complex biochemical reactions, including oxidation of proteins and lipids. The combined effects may cripple the cells beyond repair. This communication will focus on the thermal effects of electric shock. After a brief review of the current state of knowledge on thermal denaturation of soluble enzymes and muscle proteins, this paper will describe experiments on the thermal denaturation of cellular components and functions, such as nucleosomes, and the electron transport chain and ATP synthetic enzymes of the mitochondrial inner membranes. Data will show that lipid peroxidation and the subsequent loss of the energy-transducing ability of the cells may occur even at moderate temperatures between 40 degrees C and 45 degrees C. However, lipid peroxidation may be prevented with reducing reagents such as mercaptoethanol, dithiothreitol, and ascorbic acid. Reactivation of denatured cellular proteins and functions may also be possible and a strategy for doing so is discussed.


Transformation of plasmid DNA into E. coli using the heat shock method

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 plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42 degrees C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37 degrees C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.


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

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 plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42ଌ for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37ଌ for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.


Methods of Gene Transfer: 6 Methods

This article throws light upon the six methods of gene transfer. The six methods are: (1) Transformation (2) Conjugation (3) Electroporation (4) Liposome-Mediated Gene Transfer (5) Transduction and (6) Direct Transfer of DNA.

Method # 1. Transformation:

Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The uptake of plasmid DNA by E.coli is carried out in ice-cold CaCl2 (0-5°C), and a subsequent heat shock (37-45°C for about 90 sec). By this technique, the transformation frequency, which refers to the fraction of cell population that can be transferred, is reasonably good e.g. approximately one cell for 1000 (10 -3 ) cells.

Transformation efficiency:

It refers to the number of trans-formants per microgram of added DNA. For E.coli, transformation by plasmid, the transformation efficiency is about 10 7 to 10 8 cells per microgram of intact plasmid DNA. The bacterial cells that can take up DNA are considered as competent. The competence can be enhanced by altering growth conditions.

The mechanism of the transformation process is not fully understood. It is believed that the CaCI2 affects the cell wall, breaks at localized regions, and is also responsible for binding of DNA to cell surface. A brief heat shock (i.e. the sudden increase in temperature from 5°C to 40°C) stimulates DNA uptake. In general, large-sized DNAs are less efficient in transforming.

Other chemical methods for transformation:

Calcium phosphate (in place of CaCI2) is preferred for the transfer of DNA into cultured cells. Sometimes, calcium phosphate may result in precipitate and toxicity to the cells. Some workers use diethyl amino ethyl dextran (DEAE -dextran) for DNA transfer.

Method # 2. Conjugation:

Conjugation is a natural microbial recombination process. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer single-stranded DNA (from donor to recipient). Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids).

Conjugation can occur among the cells from different genera of bacteria (e.g Salmonella and Shigella cells). This is in contrast to transformation which takes place among the cells of a bacterial genus. Thus by conjugation, transfer of genes from two different and unrelated bacteria is possible.

The natural phenomenon of conjugation is exploited for gene transfer. This is achieved by transferring plasmid-insert DNA from one cell to another. In general, the plasmids lack conjugative functions and therefore, they are not as such capable of transferring DNA to the recipient cells. However, some plasmids with conjugative properties can be prepared and used.

Method # 3. Electroporation:

Electroporation is based on the principle that high voltage electric pulses can induce cell plasma membranes to fuse. Thus, electroporation is a technique involving electric field-mediated membrane permeabilization. Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by electric pulses) from the suspending solution.

Electroporation is a simple and rapid technique for introducing genes into the cells from various organisms (microorganisms, plants and animals).

The basic technique of electroporation for transferring genes into mammalian cells is depicted in Fig. 6.11. The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.

Electroporation is an effective way to transform E.coli cells containing plasmids with insert DNAs longer than 100 kb. The transformation efficiency is around 10 9 transformants per microgram of DNA for small plasmids (about 3kb) and about 10 6 for large plasmids (about 130 kb).

Method # 4. Liposome-Mediated Gene Transfer:

Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids. Several techniques have been developed to encapsulate DNA in liposomes. The liposome- mediated gene transfer, referred to as lipofection, is depicted in Fig. 6.12.

On treatment of DNA fragment with liposomes, the DNA pieces get encapsulated inside liposomes. These liposomes can adher to cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. The positively charged liposomes very efficiently complex with DNA, bind to cells and transfer DNA rapidly.

Lipofection is a very efficient technique and is used for the transfer of genes to bacterial, animal and plant cells. T

Method # 5. Transduction:

Sometimes, the foreign DNA can be packed inside animal viruses. These viruses can naturally infect the cells and introduce the DNA into host cells. The transfer of DNA by this approach is referred to as transduction.

Method # 6. Direct Transfer of DNA:

It is possible to directly transfer the DNA into the cell nucleus. Microinjection and particle bombardment are the two techniques commonly used for this purpose.

DNA transfer by microinjection is generally used for the cultured cells. This technique is also useful to introduce DNA into large cells such as oocytes, eggs and the cells of early embryos. The term transfection is used for the transfer DNA into eukaryotic cells, by various physical or chemical means.


Heat shock vs electroporation - Biology

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ELECTROPORATION OF EMBRYOGENIC PROTOPLASTS OF SWEET ORANGE ( CITRUS SINENSIS (L.) OSBECK) AND REGENERATION OF TRANSFORMED PLANTS

RANDALL P. NIEDZ, 1,2 W. L. McKENDREE, 1 R. G. SHATTERS Jr. 1

1 Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Ft. Pierce, FL 34945-3030
2 Author to whom correspondence should be addressed: [email protected]

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Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet orange [ Citrus sinensis (L.) Osbeck cv. Hamlin]. Electric field strength (375–450 V cm −1 ), vector DNA concentration (100 μg ml −1 ), carrier DNA concentration (100 μg ml −1 ), electroporation buffer (pH 8), and pre-electroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24 h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined, GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg −1 (protein) min −1 . Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.

RANDALL P. NIEDZ , W. L. McKENDREE , and R. G. SHATTERS Jr. "ELECTROPORATION OF EMBRYOGENIC PROTOPLASTS OF SWEET ORANGE ( CITRUS SINENSIS (L.) OSBECK) AND REGENERATION OF TRANSFORMED PLANTS," In Vitro Cellular and Developmental Biology - Plant 39(6), 586-594, (1 November 2003). https://doi.org/10.1079/IVP2003463

Received: 4 February 2003 Accepted: 1 May 2003 Published: 1 November 2003

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Essay: Gene transfer techniques – pros and cons

Gene transfer is a technique to stably and efficiently introduce functional genes (that are usually cloned) into the target cells. Genetic transfer is the mechanism by which DNA is transferred from a donor to a recipient. Once donor DNA is inside the recipient, crossing over can occur. The result is a recombinant cell that has a genome different from either the donor or the recipient. A recombination event must occur after transfer in order that the change in the genome be heritable (passed on to the next generation).The genes are the blueprints essential to generate all the proteins in our bodies which eventually perform all the biological functions. Therefore, when a gene is efficiently introduced into a target cell of the host, the protein which is encoded by that gene is produced .
Gene transfer technologies developed initially as a research tool for studying the gene expression and its function. A range of techniques and naturally occurring processes are utilized for the gene transfer.
Techniques are available for the introduction of DNA into many different cell types in culture, either to study gene function and regulation or to produce large amounts of recombinant protein.
Cell cultures have therefore been used on a commercial scale to synthesize products such as antibodies, hormones, growth factors, cytokines and viral coat proteins for immunization.The most important application of this technology is in vivo gene therapy, i.e. the introduction of DNA into the cells of live animals in order to treat disease. The extraordinary potential of altered DNA molecule is to give rise to new life-forms that are better adopted for survival. Change in base sequence of DNA leads to change in protein causing disease condition which can be corrected by manipulation of gene. Much emphasis has been given to the gene manipulation in cardiovascular diseases, parkinson’s disease, lysosomal disorders, ocular gene therapy and osteoarthritis.
Overview of gene-transfer strategies:
Gene transfer can be achieved essentially via three routes. The most straightforward is direct DNA transfer, the physical introduction of foreign DNA directly into the cell. For example, in cultured cells this can be done by microinjection, whereas for cells in vivo direct transfer is often achieved by bombardment with tiny DNA-coated metal particles. The second route is termed transfection, and this encompasses a number of techniques, some chemical and some physical, which can be used to persuade cells to take up DNA from their surroundings. The third is to package the DNA inside an
animal virus, since viruses have evolved mechanisms to naturally infect cells and introduce their own nucleic acid. The transfer of foreign DNA into a cell by this route is termed transduction.
The choice of methods of DNA transfer depends upon the target cells in which transformation will be performed. It also depends upon the objectives of gene manipulation. The transfection may be either stable or transient. Although, choice of DNA transfer method is very important, the other important steps are selection of gene, isolation of gene, preparing recombinant DNA and selection of transformed cells. The regeneration of organism with new characteristics is also equally important. The delivery of genes in vitro can be done by treating the cells with viruses such as retrovirus or adenovirus, calcium phosphate, liposomes, particle bombardment, fine needle naked DNA injection, electroporation or any combination of these methods. These are the powerful tools for research and have possible applications in gene therapy.
FACTORS TO CONSIDER WHEN SELECTING A GENE DELIVERY METHOD
The advancement of new technologies has made gene delivery both more efficient yet more confusing given the formidable array of reagents and methods now available. Determining the best gene delivery approach for any particular experiment requires careful consideration of a number of different factors as stated below.
1. Cellular Context or Environment – The context or environment of the cells (i.e., in vivo vs. in
vitro vs. ex vivo) to which nucleic acids will be delivered is the first factor to consider when
deciding upon a gene transfer method. The obstacles and challenges posed by delivery to in
vitro cell cultures are very different than those posed by in vivo or ex vivo cells, and require
different tools and techniques for overcoming them. For example, targeting nucleic acids to
particular cells while avoiding other cells is usually a major concern for in vivo gene transfer
applications, sometimes a concern for ex vivo applications, but usually not a concern for in vitro
applications.
Another important issue that must be addressed when planning in vivo (but not in vitro) gene
delivery studies is the immunogenicity of the gene transfer system. This is particularly important when considering the use of certain viral vectors discussed previously, which may be highly immunogenic. Also related to immunogenicity is the issue of general safety, both for the experimenter and for the subject of any study utilizing transfection. Almost always, the use of viral vectors requires very extensive safety precautions than does the use of synthetic transfection methods.
2. Cell or Tissue Type – The origin of the cells or tissue to which nucleic acids will be
transferred is typically the second parameter considered when selecting a gene delivery
method. For a number of different reasons, the difficulty of cellular nucleic acid delivery varies
from cell type to cell type. Because of this, it is always important to review the literature to
determine if the cell type of interest has previously been utilized for gene transfer experiments,
which exact methods have been used, and what delivery efficiencies were achieved, i.e., what
percentages of cells actually transfected or transduced, with each method.
Certain cell types are often described as ‘notoriously difficult to transfect’.
These include primary cell cultures of many varieties. Successful gene transfer to such cells
usually requires a good deal of research before selecting any particular gene delivery method.
3. Delivery Efficiency – For most applications, transfecting or transducing as many cells as
possible is preferred, especially when it is necessary to detect the presence of rare transgene
products. Thus, it is important to determine the level of delivery efficiency required for a given
application before deciding on a particular gene transfer method. In most cases, it is found that
viral vectors provide the highest delivery efficiencies. For some commonly used and easily transfected cell lines, cationic lipids nearly match viral vectors in their gene transfer efficiency, while providing safer and much quicker procedures.
4. Stable vs. Transient Gene Delivery – Gene transfer procedures are frequently categorized
by whether the delivered gene remains separate from the host cell chromosome or whether it is integrated into the host cell chromosome. In the first case, known as transient gene delivery, expression of the transgene typically dissipates after a given period of time – usually within
several days – because the expression vector is either degraded or expelled from the host cell.
In the second case, known as stable gene delivery, expression of the transferred gene is
prolonged, or stable, because the vector is integrated into the host cell chromosome. Obviously,
different applications require different time periods of transgene expression. Thus, a careful
comparison between different gene delivery methodologies will allow a suitable choice for
generating the desired transient or stable expression.
In the case of viral vectors, determining whether a particular virus is suited for stable or transient
transduction is simply a matter of learning the general characteristics of the vector. In the case
of chemical or physical/mechanical gene transfer, determining whether a given method is
suitable for transient vs. stable delivery may be as easy as reading sales literature from the
manufacturer or checking primary research literature.
5. Type of Delivered Molecule – Another factor to consider when selecting a gene delivery
method is what type of nucleic acid or other molecule is being transfected or transduced. For
example, a reagent that efficiently delivers plasmids may not efficiently deliver DNA
oligonucleotides. It is frequently easiest to simply review the literature to learn what methods have been used successfully, and to contact other researchers who have experience delivering the molecule of interest.
6. Cytotoxicity – Another issue that comes up frequently when choosing among different gene
delivery methods is the cytotoxicity of the various methods and reagents. Some methods, such
as electroporation and gene guns, can be extremely harsh on cells and often result in the death
of a majority of cells. Also, the DEAE-dextran can be quite harsh on some cell types, especially
when a DMSO or glycerol shock step is used as part of the procedure. In some applications,
such as when a small number of transfectants are required from a recalcitrant cell type, this is
not a problem. In the majority of applications however, a high percentage of cell death is simply
not acceptable. The plethora of available cationic lipids and polymer reagents have varying
degrees of toxicity with different cell types, and their concentrations during transfection can be
optimized as needed to minimize cell death.
7. Suspension vs. Adherent cells – Most commonly transfected cells grow – and are
transfected – while attached to some type of support, whether that support is the tissue from
which they are derived, some inert membrane or scaffolding, or the surface of a tissue culture
plate. Less common are cells that grow while suspended in medium, such as those derived from
blood and other bodily fluids. Suspension cells are generally more difficult to transfect
therefore, it may be necessary to rely on harsher gene delivery methods, such as
electroporation, gene guns, microinjection, or viral vectors for acceptable transfection levels.
Also, some cationic lipids can effectively transfect suspension cells, and should be tried when
possible due to their simplicity and cost-effectiveness.
8. Expertise – The degree of difficulty in performing the various transfection techniques can
vary significantly, and therefore the expertise and experience of the researcher is usually an
important factor to consider. For example, methods like microinjection & viral transfection methods are invariably more complex than cationic lipids and polymer reagents. Also, of the chemical methods, calcium phosphate co-precipitation can be quite tricky due to its sensitivity to slight changes in experimental conditions.
9. Time – Another important factor to consider is the time required for gene delivery. Viral
methods, while highly efficient, can be quite time consuming, typically taking anywhere from two weeks to one month to complete. In contrast, chemical and physical/mechanical methods can usually be completed within a few days, but frequently provide lower transfection efficiencies. Thus, it is not uncommon to have to weigh the benefits of faster results and less labor against the benefit of higher efficiency.
10 Cost – Finally, cost can be a major factor when deciding on the best gene delivery
method for a given application. The most expensive methods tend to be the
physical/mechanical methods, such as electroporation and gene guns, due to the specialized
equipment they require.Viral gene delivery is perhaps the second most expensive method, since it requires the vector reagents, extensive time and labor to complete lengthy vector preparation procedures, as well as proper disposal of viral waste materials.
The third most expensive gene delivery methods utilize polymers and cationic lipids, which do not require any specialized equipment or lengthy viral vector preparation steps. Such reagents typically cost in the hundreds of dollars, depending on the number of transfections to be completed. Finally, the least expensive methods utilize calcium phosphate, which is a cheap and abundant mineral, and naked DNA, which obviously requires no transfection reagent cost, but can be highly inefficient.
GENE TRANSFER TECHNIQUES
TRANSFORMATION
Transformation is the naturally occurring process of gene transfer which involves absorption of the genetic material by a cell through cell membrane causing the fusion of the foreign DNA with the native DNA resulting in the genetic expression of the received DNA. Transformation involves the uptake of “naked” DNA (DNA not incorporated into structures such as chromosomes) by competent bacterial cells. Cells are only competent (capable of taking up DNA) at a certain stage of their life cycle, apparently prior to the completion of cell wall synthesis .
Transformation is usually a natural method of gene transfer but as a result of technological advancement originated the artificial or induced transformation. Thus there are two types called as natural transformation and artificial or induced transformation. In natural transformation, the foreign DNA attaches itself to the host cell DNA receptor and with the help of the protein DNA translocase it enters the host cell. The presence of nucleases restricts the entry of two strands of the DNA, destroys a single strand thus allowing only one strand to enter the host cell. Any DNA that is not integrated into the chromosome will be degraded. This single stranded DNA mingles with the host genetic material successfully.
The artificial or induced method of transformation is done under laboratory condition which is either a chemical mediated gene transfer or done by electroporation.. Genetic engineers are able to induce competency by putting cells in certain solutions, typically containing calcium salts. At the entry site, endonucleases cut the DNA into fragments of 7,000-10,000 nucleotides, and the double-stranded DNA separates into single strands. The single-stranded DNA may recombine with the host’s chromosome once inside the cell. This recombination replaces the gene in the host with a variant – albeit homologous – gene.
Drawbacks:
DNA from a closely related genus may be acquired but, in general, DNA is not exchanged between distantly related microbes.
Not all bacteria can become competent.
CONJUGATION
Conjugation is a means of gene transfer in many species of bacteria. Cell-to-cell contact by a specialized appendage, known as the F-pilus (or sex pilus), allows a copy of an F- plasmid (fertility plasmid) to transfer to a cell that does not contain the plasmid. On rare occasions an F-plasmid may become integrated in the chromosome of its bacterial host, generating what is known as an Hfr (high frequency of recombination) cell. Such a cell can also direct the synthesis of a sex pilus. As the chromosome of the Hfr cell replicates it may begin to cross the pilus so that plasmid and chromosomal DNA transfers to the recipient cell. Such DNA may recombine with that of its new host, introducing new gene variants. Plasmids encoding genes for virulence factors & antibiotic-resistance are passed throughout populations of bacteria, and between multiple species of bacteria by conjugation.
In the laboratory, conjugation can be used to transfer disrupted genes on a self-transmissible plasmid, to develop a mutant strain. A gene of interest from a recipient E. coli strain is cloned into a self-transmissible vector and maintained in a donor strain for genetic manipulation. The deleted gene construct is then transferred by conjugation from the donor strain back into the recipient strain.
TRANSDUCTION
Transduction is another method for transferring genes from one bacterium to another the transfer is mediated by bacteriophages. A bacteriophage infection starts when the virus injects its DNA into a bacterial cell. The bacteriophage DNA may then direct the synthesis of new viral components assembled in the bacterium. Bacteriophage DNA is replicated and then packaged within the phage particles. Early in the infective cycle the phage encodes an enzyme that degrades the DNA of the host cell. Some of these fragments of bacterial DNA are packaged within the bacteriophage particles, taking the place of phage DNA. As the number of phages increases it breaks open (lyse) the cell. When released from the infected cell, a phage that contains bacterial genes can continue to infect a new bacterial cell, transferring the bacterial genes.
Sometimes genes transferred in this manner become integrated into the genome of their new bacterial host by homologous recombination. Such transduced bacteria are not lysed because they do not contain adequate phage DNA for viral synthesis and these phages can enter an alternate life cycle called lysogeny. In this cycle, all the virus’s DNA becomes integrated into the genome of the host bacterium. The integrated phage, called a prophage, can confer new properties to the bacterium.
Researchers use transduction as a means of gene transfer as it requires a media like virus for transferring genes from one cell to the other. Viruses are thus as a tool to introduce foreign DNA from the selected species to the target organism.
There are two types of transduction called as generalized transduction in which any of the bacterial gene is transferred via the bacteriophage to the other bacteria and specialized transduction involves transfer of limited or selected set of genes.
Viral vectors for gene transfer:
The use of viruses as vectors for transduction, i.e. the introduction of genes into animal cells by exploiting the natural ability of the virus particle, within which the transgene is packaged, to adsorb to the surface of the cell and gain entry is being employed . Due to the efficiency with which viruses can deliver their nucleic acid into cells and the high levels of replication
and gene expression it is possible to achieve, viruses have been used as vectors not only for gene expression in cultured cells but also for gene transfer to living animals. Four classes of viral vector have been developed for use in human gene therapy and have reached phase 1 clinical trials. These are the retrovirus, adenovirus, herpesvirus and adenoassociated virus (AAV) vectors . Before introducing the individual vector systems,we discuss some general properties of viral transduction vectors. Transgenes may be incorporated into
viral vectors either by addition to the whole genome or by replacing one or more viral genes. This is generally achieved either by ligation (many viruses have been modified to incorporate unique restriction sites) or homologous recombination.
For many applications, it is favourable to use vectors from which all viral coding sequences have been deleted.These amplicons (also described as ‘gutless vectors’) contain just the cis-acting elements required for packaging and genome replication. The advantage of such vectors is their high capacity for foreign DNA and the fact that, since no viral gene products are made, the vector has no intrinsic cytotoxic effects. The choice of vector depends on the particular properties of the virus and the intended host, whether transient or stable expression is required and how much DNA needs to be packaged. For example, icosahedral viruses such as adenoviruses and retroviruses package their genomes into preformed capsids, whose volume defines the maximum amount of foreign DNA that can be accommodated. Conversely, rod-shaped viruses such as the baculoviruses form the capsid around the genome, so there are no such size constraints. There is no ideal virus for gene transfer ‘ each has its own advantages and disadvantages. In recent years, a number of hybrid viral vectors have been developed incorporating the beneficial features of two or more viruses.
Advantages:
1) viral vectors that especially efficient at transducing the intended cell type but not other cell types.
2) retroviruses are particularly proficient at delivering genes to dividing cells, such as cancer cells or immortalized cell lines
Limitations:
1) cannot deliver genes to terminally differentiated cells
2) viral vectors may be highly immunogenic
TRANSFECTION BY CHEMICAL METHODS
One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene is transfection. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. When transformation is carried out in eukaryotic cells it is termed as transfection.
1) Calcium phosphate mediated DNA transfer
The process involves a mixture of isolated DNA (10-100ug) with solution of
calcium chloride and potassium phosphate. . When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface .Cells are then incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by a process not entirely understood but thought to be endocytosis.
Transfection efficiencies using calcium phosphate can be quite low, in the range of 1-2 % . It can
be increased if very high purity DNA is used and the precipitate allowed to form slowly.
Limitations
1. Frequency is very low.
2. Integrated genes undergo substantial modification.
3. Many cells do not like having the solid precipitate adhering to them
and the surface of their culture vessel.
4. Integration with host cell chromosome is random.
5, Due to above limitations transfection applied to somatic gene therapy
is limited.
Applications:
This technique is used for introducing DNA into mammalian cells. This process has been a preferred method of identifying many oncogenes.
2)DNA transfer by DAE-Dextran method:
DNA can be transferred with the help of DAE Dextran also. DAE-Dextran may be used in the
transfection medium in which DNA is present. This is a polycationic, high molecular weight substance and is convenient for transient assays. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis.
If DEAE (diethylaminoethyl )-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO)
shock, then upto 80% transformed cell can express the transferred gene.
Advantages:
The advantage of this method is that, it is cheap, simple and can be used for transient cells
which cannot survive even short exposure of calcium phosphate.
The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency.
Limitations:
Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells.
Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection.
It does not appear to be efficient for the production of stable transfectants.
For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.
Transfer of DNA by polycation-DMSO:
As calcium phosphate method of DNA transfer is reproducible and efficient but there is narrow range of optimum conditions. DNA transfer by
Another polycationic chemical,the detergent Polybrene is carried out to increase the adsorption of DNA to the cell surface followed by a brief treatment with 25-30% DMSO to increase membrane permeability and enhance uptake of DNA. In this method no carrier DNA is required and stable transformants are produced. This method works with mouse fibroblast and chick embryo.
LIPOSOME MEDIATED GENE TRANSFER
Liposomes are spheres of lipids which can be used to transport molecules into the cells. These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. They are considered as spheres of lipid bilayers surrounding the molecule to be transported and promote transport after fusing with the cell membrane.
Liposomes for use as gene transfer vehicles are prepared by adding an appropriate mix of bilayer constituents to an aqueous solution of DNA molecules. In this aqueous environment, phospholipid hydrophilic heads associate with water while hydrophobic tails self-associate to exclude water from within the lipid bilayer. This self-organizing process creates discrete spheres of continuous lipid bilayer membrane enveloping a small quantity of DNA solution. The liposomes are then ready to be added to target cells. Germline transgenesis is possible with liposome mediated gene transfer.Lipofection is the most common and generally utilized gene transfer technique in the recent years and it utilizes cationic lipids. The combined DNA and cationic lipids act instantaneously to form structures called as lipoplexes that are more complex in structure than the simple liposomes. Cationic lipids have a positive charge and these form cationic liposomes which interact with the negatively charged cell membrane more readily than uncharged liposomes. This leads to a fusion between cationic liposome and the cell surface resulting in quick entry by endocytosis and the delivery of the DNA directly across the plasma membrane. Cationic liposomes can be produced from a number of cationic lipids that are commercially available and sold as an in vitro-transfecting agent, termed lipofectin.
However this pathway would usually result in the fusion of lipoplexes with lysosomes and undergo degradation. This problem is overcome by utilizing the neutral helper lipids, which are generally included along with the cationic lipid. This allows entrapped DNA to escape the endosomes, reach the nucleus and get access to the cell’s transcriptional machinery.The endosomal structure is destroyed by increasing the osmotic pressure created by the lipids’ buffering action within the endosomes and by the fusion of the lipid with the endosomal membrane. The ability of a lipid to destroy endosomes is one of the main characteristics of a transfection reagent.
Advantages
1. Simplicity.
2. Long term stability.
3. Low toxicity.
4. Protection of nucleic acid from degradation.
5. particularly proficient at delivering genes to dividing cells, such as cancer cells or immortalized cell lines
6. cationic lipids effectively transfect suspension cells.
Limitations:
1) Cannot deliver genes to terminally differentiated cells
ELECTROPORATION
A rapid and simple technique for introducing genes into a wide variety of microbial, plant and animal cells, including E. coli, is electroporation. Electroporation uses electrical pulse to produce transient pores in the plasma membrane thereby allowing macromolecules into the cells. These pores are known as electropores which allow the molecules, ions and water to pass from one side of the membrane to another. The electropores reseal spontaneously and the cell can recover. The pores can be recovered only if a suitable electric pulse is applied .The formation of electropores depends upon the cells that are used and the amplitude and duration of the electric pulse that is applied to them. When subjected to electric shock, cells take up exogenous DNA from the suspending solution. Once inside the cell, the DNA is integrated and the foreign gene will express. A proportion of these cells become stably transformed and can be selected if a suitable marker gene is carried on the transforming DNA. The most critical parameters are the intensity and duration of the electric pulse, and these must be determined empirically for different cell types. Electric currents can lead to dramatic heating of the cells that can results in cell death. Heating effects are minimized by using relatively high amplitude, a short duration pulse or by using two very short duration pulses. However, once optimal electroporation parameters have been established, the method is simple to carry out and highly reproducible.
Many different factors affect the efficiency of electroporation, including temperature,
various electric-field parameters (voltage, resistance and capacitance), topological form of the DNA,
and various host-cell factors (genetic background, growth conditions) and post-pulse treatment.
General applications of electroporation.
1. Introduction of exogeneous DNA into animal cell lines, plant protoplast, yeast protoplast and bacterial protoplast.
2. Electroporation can be used to increase efficiency of transformation or transfection of bacterial cells.
3. Wheat, rice, maize, tobacco have been stably transformed with frequency upto 1% by this method.
4. Genes encoding selectable marker may be used to introduce genes using electroporation.
5. To study the transient expression of molecular constructs.
6. Electroporation of early embryo may result in the production of transgenic animals.
7. Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B lymphocytes can be transformed by this technique.
8. Naked DNA may be used for gene therapy by applying electroporation device on animal cells.
Advantages of electroporation.
1. Method is fast.
2. Less costly.
3. Applied for a number of cell types.
4. Simultaneously a large number of cell can be treated.
5. High percentage of stable transformants can be produced.
DRAWBACKs
1)Limited effective range of

1 cm between the electrodes.
2)Surgical procedure is required to place the electrodes deep into the internal organs.
3)High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating.
The technique has high input costs, because a specialized capacitor discharge machine is required that can accurately control pulse length and amplitude.
4) Larger numbers of cells may be required than for other methods because, in many cases, the most efficient electroporation occurs when there is up to 50% cell death.
ULTRASONICATION
Sonication is the act of applying sound energy to agitate particles in a sample. Ultrasonic frequencies (>20 kHz) are usually used, leading to the process also being known as ultrasonication . Sonoporation, or cellular sonication, is the use of these ultrasonic frequencies for modifying the permeability of the cell plasma membrane. This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process called transfection or transformation. Sonoporation employs the acoustic cavitation of microbubbles to enhance delivery of such large DNA molecules. Although the early results could achieve only 10- to 30-fold increases in transfection efficiency in vitro, technical refinements have lead to enhancements of up to several thousand fold in vitro ,sufficient to encourage ultrasonication mediated gene transfer in vivo.
Advantages:
1) It is non-invasive and well tolerated,
2)extraordinary safety record over a wide range of frequency and intensity
3)high levels of public acceptability and understanding.
there are highly sophisticated, flexible, cost-effective and readily available diagnostic and therapeutic systems that can achieve site-specific transfer of ultrasound energy almost anywhere in the body, except perhaps the lung.
4) The bioactivity of this technique is similar to, and in some cases found superior to, electroporation.
5) in vivo is relatively nontoxic.
Limitations:
1)Extended exposure to low-frequency (<MHz) ultrasound has been demonstrated to result in complete cellular death (rupturing) as well as microvascular hemorrhage and disruption of tissue structure, thus cellular viability must also be accounted for when employing this technique.
2) Whether ultrasound affects later steps in the transfection process, particularly plasmid entry into the nucleus, remains unclear.
Applications:
Sonoporation is under active study for the introduction of foreign genes in tissue culture cells, especially mammalian cells. Sonoporation is also being studied for use in targetedGene therapy in vivo, in a medical treatment scenario whereby a patient is given modified DNA, and an ultrasonic transducer might target this modified DNA into specific regions of the patient’s body.
MICROINJECTION
In microinjection DNA can be introduced into cells or protoplast with the help of very fine needles or
glass micropipettes having the diameter of 0.5 to 10 micrometer. The DNA may be directly delivered into the nucleus or in the cytoplasm. Some of the DNA injected may be taken up by the nucleus. The desired gene in the form of plasmid or alone is injected directly into the plant protoplast.
Injection into the nucleus is more efficient than that into the cytoplasm. Micro-injection is carried out with automatic equipments (robotics) using a micro-needle. Computerized control of holding pipette, needle, microscope stage and video technology has improved the efficiency of this technique.
Advantages of microinjection.
1. Frequency of stable integration of DNA is far better as compare to other methods.
2. Method is effective in transforming primary cells as well as cells in established cultures.
3. The DNA injected in this process is subjected to less extensive modifications.
4. Mere precise integration of recombinant gene in limited copy number can be obtained.
Limitations
1. Costly.
2. Skilled personal required.
3.Has not been successful for many plant cells .
4. Embryonic cells preferred for manipulation.
5. Knowledge of mating timing, oocyte recovery is essential.
6. Method is useful for protoplasts and not for the walled cells.
7. Process causes random integration.
8. Rearrangement or deletion of host DNA adjacent to site of integration are common.
9. The technique is laborious, technically difficult, and limited to the number of cells actually injected.
Applications of microinjection.
1. Process is applicable for plant cell as well as animal cell but more common for animal cells.
2. Technique is ideally useful for producing transgenic animal quickly.
3. Procedure is important for gene transfer to embryonic cells.
4. Applied to inject DNA into plant nuclei.
5. Method has been successfully used with cells and protoplast of
tobacco, alfalfa etc.
Fig: microinjection in mouse egg.
BIOLISTICS
(MICROPROJECTILE/GENE GUN)
Biolistics or particle bombardment is a physical method that uses accelerated microprojectiles to
deliver DNA or other molecules into intact tissues and cells. Biolistics transformation is relatively new and novel method amongst the physical methods for artificial transfer of exogenous DNA. The nucleic acid is delivered through membrane penetration at a high velocity, usually connected to microprojectiles like microscopic particles of tungsten or gold coated into cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the particles pass through the cell, the DNA becomes free to integrate into the plant-cell genome.
The gene gun is a device that literally fires DNA into target cells . The DNA to be transformed
into the cells is coated onto microscopic beads made of either gold or tungsten. Beads are carefully
coated with DNA. The coated beads are then attached to the end of the plastic bullet and loaded into
the firing chamber of the gene gun. An explosive force fires the bullet down the barrel of the gun
towards the target cells that lie just beyond the end of the barrel. When the bullet reaches the end of
the barrel it is caught and stopped, but the DNA coated beads continue on toward the target cells.
Some of the beads pass through the cell wall into the cytoplasm of the target cells. Here the bead and
the DNA dissociate and the cells become transformed. Once inside the target cells, the DNA is
solubilised and may be expressed.
Applications
1. Biolistics technique has been used successfully to transform soyabean,
cotton, spruce, sugarcane, papaya, corn, sunflower, rice, maize, wheat,
tobacco etc.
2. Genomes of subcellular organelles have been accessible to genetic
manipulation by biolistic method.
3. Mitochondria of plants and chloroplast of chlamydomonas have been
transformed.
4. Method can be applied to filamentous fungi and yeast (mitochondria).
5. The particle gun has also been used with pollen, early stage
embryoids, meristems somatic embryos, plant cells, root section, seeds and pollen.
6. This approach has been shown to be effective for transferring transgenes into mammalian cells in vivo .
Advantages
1. Requirement of protoplast can be avoided. Unlike electroporation and microinjection, this technique does not require protoplasts or even single-cell isolations and is applicable to even intact plant tissue
2. Walled intact cells can be penetrated.
3. Manipulation of genome of subcellular organelles can be achieved.
4. This is also a highly mechanized or robotic mediated technique in which the
speed of the micro-projectile particles is controlled by foolproof mechanisms
5) It is also gaining in use as a method for transferring DNA construct into whole animals.
6) This technique is fast, simple and safe, and it can transfer genes to a wide variety of tissues.
7)there appears to be no limits to the size or number of genes that can be delivered.
Limitations
1. Integration is random.
2. Requirement of equipments.
3.It can be a challenge to obtain a sufficient number of cells modified by this method to see a biologically significant effect.
MICROLASER
A new technique is presented to incorporate exogeneous gene materials (DNA) into cells with a microbeam irradiation from an uv pulsed laser. A frequency-multiplied Laser, 355 m wavelength, 5 ns pulse duration, punches a self-healing hole of submicrometer aperture in cell membrane under selected irradiation conditions. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously . It takes a fraction of a second for the aperture to close, long enough to allow the foreign DNA, contained in the medium, to slip into the cell.
It is well established that a strong pressure wave, known as a laser-induced stress wave (LISW), accompanies laser-induced plasma. We have extended their method to deliver macromolecules, such as genes. Plasmid DNA (circular DNA residing in bacterial cytoplasm) is used as a vector of the gene of interest. We inject the plasmid into target tissue, on which a laser target is placed, and subject the target to irradiation with a high-intensity, nanosecond laser pulse to induce plasma and hence an LISW. By placing optically transparent material on the target, the plasma is confined, resulting in an increase in the LISW’s impulse. Interaction of tissue cells with the LISW allows plasmid to enter the cytoplasm
Advantages
1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances
(2) it is a very mild treatment virtually suitable for any cell type and
(3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).
4) . it is rapidly replacing virus mediated techniques as they have serious side effects caused by immune response and limited targeting characteristics rendering them inappropriate for clinical application
5) The method offers a clear advantage over existing methods: increases the success rate of DNA transfection as well as the efficiency of cell modification by orders of magnitude.
6) . It enables minimally invasive tissue interaction.
CONCLUSION
Thus there are a number of ways by which the genes can be introduced into the cells. With the advent of molecular tools and technologies it is now comparatively easy to introduce gene into cells without losing its integrity and biological activity. Moreover the recent development in molecular biology has made the transfer of gene with great accuracy to the target cell. The transfer of gene through different gene transfer technologies has cured a number of diseases. Research is on progress to cure those diseases which cannot be cured by using drugs. Moreover the treatment of diseases by gene transfer provides better result for a prolong period of time. It is the need of hour to discover new and cheap method of gene transfer technologies so to make the treatment of the diseases a little easier and affordable. Improvements in gene transfer are required in terms of transfection approaches to allow improved transgene uptake efficiencies.
REFERENCES
1)Society of Applied Sciences.
Gene Transfer Technologies and their Applications: K.H.KHAN
Medical Biotechnology Division, School of Biosciences and Technology,
VIT University, Vellore-632014, Tamil Nadu, India.
2)Principles of gene manipulation: R.W.Old ,S.B. Primrose and R.M.Twymann ‘ sixth edition,chapter 10 and 11.
3) BIOTECHNOLOGY 2020-From the Transparent Cell to
the Custom-Designed Process–Prof. Gerhard Kreysa Dr.-Ing. E.h. Dr.h.c. & Dr. R??diger Marquardt
4) INTRODUCTION TOBIOTECHNOLOGY AND GENETIC ENGINEERING
-A.J. NAIR, PH.D.
5)From genes to genomes- concepts and applications of DNA technology–Jeremy W Dale, Malcom von Schantz
6) http://www.nature.com
7)www.learner.org

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Heat shock vs electroporation - Biology

Article Summary:

Definition:-
Electroporation is the process of biotechnology to pass the electric current through the living surface fro example, a cell or a molecule. Through this way, pores appear in the surface of the living structure and biological material can pass through it easily. This method is usually used to enter the viruses or plasmids in the form of vectors or plasmids into the cell through pores in the cell membrane.

What is Electroporation:-
The technique of Electroporation is mostly used in the field of molecular biology. When this technique is applied on any cell by using electrical current from an external source, the cell membrane becomes more permeable allowing foreign objects enter into it. When the scientists have to insert a molecular probe, small piece of DNA or any drug which can change the function of the cell, they use this technique.

It is not easy to make pores in the cell membrane until it is exposed to proper electric field which can allow the plasma membrane to cross its dielectric strength. If the whole experiment is well controlled then after sometime, the pores of the plasma membrane can reseal, but during that time the molecules or foreign objects can enter the cell's cytoplasm. It is harmful for the cell if it is exposed to the electric current for long period of time. It results in the cell death or apoptosis.

Process of Electroporation is used mostly for the transformation of bacteria, plant protoplasts and yeast. Bacterial cell wall is made up of peptidoglycan and its derivatives. It has pores in its cell wall naturally, so when the plasmids have to enter into the bacterial cell, a small amount of electric current is used for this purpose just to let the plasmid enter into the bacterial cell. The whole process should be well controlled so that it can be observed that the bacterial cells can divide into new daughter cells containing the plasmids. This process is more effective than the chemical Electroporation. This process can also be used for the tissue culture cells to enter the foreign genes into the mammalian cells mainly.

How does process of Electroporation works?
Special kinds of devices are used for performing the process of Electroporation called as electroporators. This device is passed through the solution of the cell which contains usually bacteria but other call types can also be used for this purpose. The cell solution is put into the glass or plastic cuvette. A glass or plastic cuvette is a small circular tube which is specially designed to keep the samples for experiments of spectroscopic. It contains two aluminum electrodes one on each side. If the bacterial cell is used in the process of Electroporation then the suspension used must be of 50 microliters. Plasmids are placed in the suspension before the process starts and the whole suspension is inserted into the glass cuvette. The voltage is set to 240 volts in the electroporators and cuvette is placed inside the elctroporator. Before incubation, I milliliter of liquid medium is inserted into the cuvette and then incubation is started. After an hour of incubation, the whole solution is placed on the agarose gel. If the plasmid solution id pure then there are chances of getting better results of the experiment, otherwise bacterial cells might die due to the over explosion and process might be started once again.

Applications:-
Electroporation process of Electroporation is applicable in the field of medicine to treat the cancer and heart diseases by inserting new genes into the cancerous cell. Some other diseases can also be cured with the help of Electroporation in which there is need of tissue removal.

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Principle

Transformation process allows a bacterium to take up genes from its surrounding environment that is transformation involves the direct uptakes of fragments of DNA by a recipient cell and the acquisition of new genetic characteristics. There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. The second major parameter is the genetic constitution of the host strain of the organism being transformed. Competent cells are capable of uptaking DNA from their environment and expressing DNA as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Calcium chloride treatment is one of the best methods for the preparation of competent cells. Competence results from alterations in the cell wall that makes it permeable to large DNA molecules. This is a naturally occurring process and through this bacteria can transfer advantageous characteristics, such as antibiotic resistance. Bacteria can take DNA from the environment in the form of plasmid. Most of them are double stranded circular DNA molecules and many can exist at very high copy numbers within a single bacterial cell. Many naturally occurring plasmids carry an antibiotic resistant gene referred to as a marker.

In the process of transformation, the competent cells are incubated with DNA in ice. Then it is placed in a water bath at 42ºC and further plunging them in ice. This process will take up the DNA into the bacterial cell. Then it is plated in an agar plate containing appropriate antibiotic. The presence of an antibiotic marker on the plasmid allows for rapid screening of successful transformants. Blue &ndashwhite selection (Alpha complementation) can be used to determine which plasmids carry an inserted fragment of DNA and which do not. These plasmids contain an additional gene (lac Z) that encodes for a portion of the enzyme &beta &ndash galactosidase. When it transformed into an appropriate host, one containing the gene for the remaining portion of &beta &ndashgalactosidase, the intact enzyme can be produced and these bacteria form blue colonies in the presence of X &ndash gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside) and a gratuitous inducer called IPTG (Isopropyl &beta-D- Thiogalactopyronoside). These plasmids contains a number of cloning sites within the lac Z gene, and any insertion of foreign DNA into this region results in the loss of the ability to form active &beta &ndashgalactosidase. Therefore colonies that carry the plasmid with the insert, ie, Transformants will remain white and the colonies without the foreign DNA (Non-Transformants) will remain Blue. We can also calculate the efficiency of transformation by using the concentration of DNA and number of transformed colonies.


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