Transformation efficiency

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Transformation efficiency refers to the ability of a cell to take up and incorporate exogenous DNA, such as plasmids, during a process called transformation. The efficiency of transformation is typically measured as the number of transformants (cells that have taken up the exogenous DNA) per microgram of DNA added to the cells. A higher transformation efficiency means that more cells are able to take up the DNA, and a lower efficiency means that fewer cells are able to do so.

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In molecular biology, transformation efficiency is a crucial parameter, it is used to evaluate the ability of different methods to introduce plasmid DNA into cells and to compare the efficiency of different plasmid, vectors and host cells. This efficiency can be affected by a number of factors, including the method used for introducing the DNA, the type of cell and plasmid used, and the conditions under which the transformation is performed. Therefore, measuring and optimizing transformation efficiency is an important step in many molecular biology applications, including genetic engineering, gene therapy and biotechnology.

Measurement

By measuring the transformation efficiency, we can utilize the information from our experiment to evaluate how effectively our transformation went. This is a quantification of how many cells were altered by 1 µg of plasmid DNA. In essence, it is a sign that the transformation experiment was successful. [1] It should be determined under conditions of cell excess. [2]

Transformation efficiency is typically measured as the number of transformed cells per total number of cells. It can be represented as a percentage or as colony forming units (CFUs) per microgram of DNA.

One of the most common ways to measure transformation efficiency is by performing a colony forming assay. Here is an example of how to calculate transformation efficiency using colony forming units (CFUs): [3]

  1. Plate a known number of cells on agar plates containing the appropriate antibiotics.
  2. Incubate the plates for a period of time (usually overnight) at the appropriate temperature and conditions for the cells.
  3. Count the number of colonies that grow on the plates. This represents the number of cells that have taken up and expressed the plasmid DNA.
  4. To calculate the transformation efficiency, divide the number of colonies by the number of cells plated and multiply by 100. The result will be the transformation efficiency as a percentage.

For example, if you plate 1x 107 cells and count 1000 colonies, the transformation efficiency is: (1000/1x 107) x 100 = 0.1%

Alternatively, CFUs can be reported per microgram of DNA used for the transformation. This can be calculated by multiplying the number of colonies by the volume of the culture plated and dividing by the amount of DNA used.

Quantitative PCR (qPCR) - This method utilizes the fact that the plasmid DNA will have a specific gene or sequence that is not present in the host cell genome, and therefore can be used as a target for qPCR. By quantifying the number of copies of this specific gene or sequence in the transformed cells, it is possible to determine the amount of plasmid DNA present in the cell, and thus the transformation efficiency. [4]

Fluorescent assay - This method relies on the use of a plasmid that contains a fluorescent protein or reporter gene. The transformed cells are then analyzed by flow cytometry or fluorescence microscopy to determine the number of cells that express the fluorescent protein. The transformation efficiency is then calculated as the percentage of cells that express the fluorescent protein. [5]

The number of viable cells in a preparation for a transformation reaction may range from 2×108 to 1011; most common methods of E. coli preparation yield around 1010 viable cells per reaction. The standard plasmids used for determination of transformation efficiency in Escherichia coli are pBR322 or other similarly sized or smaller vectors, such as the pUC series of vectors. Different vectors however may be used to determine their transformation efficiency. 10–100 pg of DNA may be used for transformation, more DNA may be necessary for low-efficiency transformation (generally saturation level is reached at over 10 ng). [6]

After transformation, 1% and 10% of the cells are plated separately, the cells may be diluted in media as necessary for ease of plating. Further dilution may be used for high efficiency transformation.

A transformation efficiency of 1×108 cfu/μg for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being introduced into cells. In E. coli, the theoretical limit of transformation efficiency for most commonly used plasmids would be over 1×1011 cfu/μg. In practice the best achievable result may be around 2–4×1010 cfu/μg for a small plasmid like pUC19, and considerably lower for large plasmids.

Factors affecting transformation efficiency

Individual cells are capable of taking up many DNA molecules, but the presence of multiple plasmids does not significantly affect the occurrence of successful transformation events. [7] A number of factors may affect the transformation efficiency: [2]

Plasmid size – A study done in E. coli found that transformation efficiency declines linearly with increasing plasmid size, i.e. larger plasmids transform less well than smaller plasmids. [7] [8] [9]

Forms of DNA – Supercoiled plasmid have a slightly better transformation efficiency than relaxed plasmids – relaxed plasmids are transformed at around 75% efficiency of supercoiled ones. [7] Linear and single-stranded DNA however have much lower transformation efficiency. Single-stranded DNAs are transformed at 104 lower efficiency than double-stranded ones.

Media composition – The composition of the media used in the transformation process can affect the efficiency. For example, certain media supplements can increase the natural competence of cells. [10]

Genotype of cells – Cloning strains may contain mutations that improve the transformation efficiency of the cells. For example, E. coli K12 strains with the deoR mutation, originally found to confer an ability of cell to grow in minimum media using inosine as the sole carbon source, have 4-5 times the transformation efficiency of similar strains without. For linear DNA, which is poorly transformed in E. coli, the recBC or recD mutation can significantly improve the efficiency of its transformation. [11]

Culture conditions – E. coli cells are more susceptible to be made competent when it is growing rapidly, cells are therefore normally harvested in the early log phase of cell growth when preparing competent cells. The optimal optical density for harvesting cells normally lies around 0.4, although it may vary with different cell strains. A higher value of 0.94-0.95 has also been found to produce good yield of competent cells, but this can be impractical when cell growth is rapid. [12]

Presence of antibiotics – The presence of antibiotics can increase the efficiency of transformation by inhibiting the growth of non-transformed cells and selecting for transformed cells that are resistant to the antibiotic. For instance, the use of β-lactam antibiotics has been shown for glutamate-producing bacteria to increase it's transformation efficiencies. [13] [14] [15]

Plasmid origin of replication – The origin of replication of the plasmid used in the transformation process can affect the efficiency in several ways. The copy number of the plasmid in the cell, the activity of the origin of replication in the host cells, and the expression of the genes on the plasmid can all affect the efficiency. The plasmid with a high copy number origin of replication will generally have a higher transfection efficiency than one with a low copy number origin, using a plasmid with an origin of replication that is active in the host cell can lead to a higher transfection efficiency. [16]

Transformationconditions – The method of preparation of competent cells, the length of time of heat shock, temperature of heat shock, incubation time after heat shock, growth medium used, pH and various additives, all can affect the transformation efficiency of the cells. The presence of contaminants as well as ligase in a ligation mixture can reduce the transformation efficiency in electroporation, [17] and inactivation of ligase or chloroform extraction of DNA may be necessary for electroporation, alternatively only use a tenth of the ligation mixture to reduce the amount of contaminants. Normal preparation of competent cells can yield transformation efficiency ranging from 106 to 108 cfu/μg DNA. Protocols for chemical method however exist for making super competent cells that may yield a transformation efficiency of over 1 x 109. [18]

Damage to DNA – Exposure of DNA to UV radiation in standard preparative agarose gel electrophoresis procedure for as little as 45 seconds can damage the DNA, and this can significantly reduce the transformation efficiency. [19] Adding cytidine or guanosine to the electrophoresis buffer at 1 mM concentration however may protect the DNA from damage. A higher-wavelength UV radiation (365 nm) which cause less damage to DNA should be used if it is necessary work for work on the DNA on a UV transilluminator for an extended period of time. This longer wavelength UV produces weaker fluorescence with the ethidium bromide intercalated into the DNA, therefore if it is necessary to capture images of the DNA bands, a shorter wavelength (302 or 312 nm) UV radiations may be used. Such exposure however should be limited to a very short time if the DNA is to be recovered later for ligation and transformation.

Efficiency of transformation methods

The method used for introducing the DNA have a significant impact on the transformation efficiency. [20]

Electroporation

Electroporation tends to be more efficient than chemical methods and can be applied to a wide range of species and to strains that were previously resistant and recalcitrant to transformation techniques. [21] [22]

Electroporation has been found to have an average yield typically between 104 - 108 CFU/ug . However, a transformation efficiencies as high as 0.5-5 x 1010 colony forming units (CFU) per microgram of DNA for E. coli. For samples that are hard to handle, like cDNA libraries, gDNA, and plasmids larger than 30 kb, it is suggested to use electrocompetent cells that have transformation efficiencies of over 1 x 1010 CFU/µg. This will ensure a high success rate in introducing the DNA and forming a large number of colonies. [23] It is important to adjust and optimize the electroporation buffer (Increasing the concentration of the electroporation buffer can result in increased transformation efficiencies ) and the shape, strength, number, and number of pulses these electrical parameters play a key role in transformation efficiency. [24]

Chemical transformation

Chemical transformation or heat shock can be performed in a simple laboratory setup, typically yielding transformation efficiencies that are adequate for cloning and subcloning applications, approximately 106 CFU/µg. One of the early methods used was a combination of CaCl2 and MgCl2 to treat the cells. However, these methods resulted in transformation efficiencies, with a maximum of 105 - 106 colony forming units (CFU) per microgram of plasmid DNA. [23] Later research found that certain cations, such as Mn2+, Ca2+, Ba2+, Sr2+ and Mg2+ could have a positive effect on transformation efficiencies, with Mn2+ showing the greatest effect. [25]

Restriction barriers to an efficient transformation

Some bacterial cells have restriction-modification systems that can degrade exogenous plasmids that are foreign to the host cell. This can greatly reduce the efficiency of transformation. [26] [27] This is due to restriction systems in the recipient cells that target and destroy exogenous DNA. These systems recognize exogenous DNA based on differences in methylation patterns. To address this problem, strategies such as altering the methylation of the exogenous DNA using commercial methylases or reducing the restriction activity in the recipient cells have been applied. [28] [29] For example, using methylation-negative mutants or temporarily inactivating the restriction system with heat can reduce the recipient cell's ability to impose restrictions on the exogenous DNA. [30]

See also

Related Research Articles

<span class="mw-page-title-main">Bacterial conjugation</span> Method of bacterial gene transfer

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

<span class="mw-page-title-main">Transformation (genetics)</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 kb of DNA, limits based on the normal bacteriophage packaging size. They can replicate as plasmids if they have a suitable origin of replication (ori): for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication, or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

Triparental mating is a form of bacterial conjugation where a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second bacterial strain into a third bacterial strain. Plasmids are introduced into bacteria for such purposes as transformation, cloning, or transposon mutagenesis. Triparental matings can help overcome some of the barriers to efficient plasmid mobilization. For instance, if the conjugative plasmid and the mobilizable plasmid are members of the same incompatibility group they do not need to stably coexist in the second bacterial strain for the mobilizable plasmid to be transferred.

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<span class="mw-page-title-main">Plasmid preparation</span>

A plasmid preparation is a method of DNA extraction and purification for plasmid DNA, it is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Many methods have been developed to purify plasmid DNA from bacteria. During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.

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<span class="mw-page-title-main">Exogenous DNA</span> DNA originating from outside an organism

Exogenous DNA is DNA originating outside the organism of concern or study. Exogenous DNA can be found naturally in the form of partially degraded fragments left over from dead cells. These DNA fragments may then become integrated into the chromosomes of nearby bacterial cells to undergo mutagenesis. This process of altering bacteria is known as transformation. Bacteria may also undergo artificial transformation through chemical and biological processes. The introduction of exogenous DNA into eukaryotic cells is known as transfection. Exogenous DNA can also be artificially inserted into the genome, which revolutionized the process of genetic modification in animals. By microinjecting an artificial transgene into the nucleus of an animal embryo, the exogenous DNA is allowed to merge the cell's existing DNA to create a genetically modified, transgenic animal. The creation of transgenic animals also leads into the study of altering sperm cells with exogenous DNA.

<span class="mw-page-title-main">Blue–white screen</span> DNA screening technique

The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

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A P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of other sequences for a variety of bioengineering purposes in bacteria. It is one type of the efficient cloning vector used to clone DNA fragments in Escherichia coli cells.

Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic (bacterial) cell biology. The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher temperature (+42 degrees Celsius) for a short time.

<span class="mw-page-title-main">Plasmid-mediated resistance</span> Antibiotic resistance caused by a plasmid

Plasmid-mediated resistance is the transfer of antibiotic resistance genes which are carried on plasmids. Plasmids possess mechanisms that ensure their independent replication as well as those that regulate their replication number and guarantee stable inheritance during cell division. By the conjugation process, they can stimulate lateral transfer between bacteria from various genera and kingdoms. Numerous plasmids contain addiction-inducing systems that are typically based on toxin-antitoxin factors and capable of killing daughter cells that don't inherit the plasmid during cell division. Plasmids often carry multiple antibiotic resistance genes, contributing to the spread of multidrug-resistance (MDR). Antibiotic resistance mediated by MDR plasmids severely limits the treatment options for the infections caused by Gram-negative bacteria, especially family Enterobacteriaceae. The global spread of MDR plasmids has been enhanced by selective pressure from antimicrobial medications used in medical facilities and when raising animals for food.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

<span class="mw-page-title-main">Competence factor</span>

The ability of a cell to successfully incorporate exogenous DNA, or competency, is determined by competence factors. These factors consist of certain cell surface proteins and transcription factors that induce the uptake of DNA.

References

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