Transformation efficiency
Encyclopedia
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence
of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation
procedure. Transformants are cells which have taken up DNA (foreign, artificial or modified) and which can express genes on the introduced DNA.
are pBR322
or the smaller pUC series of vectors. The number of viable cells in a transformation reaction may range from 2x8 to 1011 (most common methods of E. coli preparation yield around 1010 viable cells per reaction). 10-100 pg of DNA may be used for transformation, more DNA may be necessary for low-efficiency transformation. After transformation, plate separately 1% and 10% of the cells (dilute cells in media as necessary for ease of plating), but further dilution may be used for high efficiency transformation.
Transformation efficiency is measured in transformants or colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1x108 cfu/μg for a small plasmid like pUC19
is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed. In E. coli, the theoretical limit of transformation efficiency for most commonly-used plasmids would be over 1x1011 cfu/μg. In practice the best achievable result may be just over 2x1010 cfu/μg for a small plasmid like pUC19, and considerably lower for large plasmids.
Plasmid size — A study done in E. coli found that transformation efficiency declines linearly with increasing plasmid
size, and that there is a similar transformation efficiency for relaxed and supercoiled
plasmids. This study also found that individual cells were capable of taking up many DNA molecules, and that the presence of multiple plasmids did not significantly affect the occurrence of successful transformation events. .
Forms of DNA — Supercoiled plasmid does not appear to have significantly better transformation efficiency than relaxed plasmids which are transformed at around 75% efficiency of supercoiled ones. Linear and single-stranded DNA however have much lower transformation efficiency. Single-stranded DNAs are transformed at 104 lower efficiency than double-stranded ones.
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, recBC or recD mutation can significant improve the efficiency of its transformation.
Growth of cells — E. coli cells are more susceptible to be made competent at a particular stage of their growth cycle, possibly when the cell volume is the greatest. When preparing competent cells, cells are therefore harvested at particular optical density (normally around 0.4, higher value of 0.94-0.95 may be used but impractical when cell growth is rapid.)
Methods of transformation — The method of preparation of competent cells, the length of time of heat shock, temperature of heat shock, 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, and heat deactivation of ligase may be necessary for electroporation. Normal preparation of compentent cells can yield transformation efficiency ranging from 106 to 108 cfu/μg DNA. Protocols for chemical method however exist for making supercompetent cells that may yield a transformation efficiency of over 109. Electroporation method in general has better transformation efficiency than chemical methods with over 1010 cfu/μg DNA possible, and it allows large plasmids of 200 kb in size to be transformed.
Competence (biology)
In microbiology, genetics, cell biology and molecular biology, competence is the ability of a cell to take up extracellular DNA from its environment...
of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
procedure. Transformants are cells which have taken up DNA (foreign, artificial or modified) and which can express genes on the introduced DNA.
Measure of transformation efficiency
Transformation effiency should be determined under conditions of cell excess. The standard plasmids used for determination of transformation efficiency in Escherichia coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
are pBR322
PBR322
pBR322 is a plasmid and was the first widely-used E. coli cloning vectors. Created in 1977, it was named after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez....
or the smaller pUC series of vectors. The number of viable cells in a transformation reaction may range from 2x8 to 1011 (most common methods of E. coli preparation yield around 1010 viable cells per reaction). 10-100 pg of DNA may be used for transformation, more DNA may be necessary for low-efficiency transformation. After transformation, plate separately 1% and 10% of the cells (dilute cells in media as necessary for ease of plating), but further dilution may be used for high efficiency transformation.
Transformation efficiency is measured in transformants or colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1x108 cfu/μg for a small plasmid like pUC19
PUC19
pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs...
is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed. In E. coli, the theoretical limit of transformation efficiency for most commonly-used plasmids would be over 1x1011 cfu/μg. In practice the best achievable result may be just over 2x1010 cfu/μg for a small plasmid like pUC19, and considerably lower for large plasmids.
Factors affecting transformation efficiency
A number of factors may affect the transformation efficiency:Plasmid size — A study done in E. coli found that transformation efficiency declines linearly with increasing plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
size, and that there is a similar transformation efficiency for relaxed and supercoiled
DNA supercoil
DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on the polymer. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to...
plasmids. This study also found that individual cells were capable of taking up many DNA molecules, and that the presence of multiple plasmids did not significantly affect the occurrence of successful transformation events. .
Forms of DNA — Supercoiled plasmid does not appear to have significantly better transformation efficiency than relaxed plasmids which are transformed at around 75% efficiency of supercoiled ones. Linear and single-stranded DNA however have much lower transformation efficiency. Single-stranded DNAs are transformed at 104 lower efficiency than double-stranded ones.
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, recBC or recD mutation can significant improve the efficiency of its transformation.
Growth of cells — E. coli cells are more susceptible to be made competent at a particular stage of their growth cycle, possibly when the cell volume is the greatest. When preparing competent cells, cells are therefore harvested at particular optical density (normally around 0.4, higher value of 0.94-0.95 may be used but impractical when cell growth is rapid.)
Methods of transformation — The method of preparation of competent cells, the length of time of heat shock, temperature of heat shock, 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, and heat deactivation of ligase may be necessary for electroporation. Normal preparation of compentent cells can yield transformation efficiency ranging from 106 to 108 cfu/μg DNA. Protocols for chemical method however exist for making supercompetent cells that may yield a transformation efficiency of over 109. Electroporation method in general has better transformation efficiency than chemical methods with over 1010 cfu/μg DNA possible, and it allows large plasmids of 200 kb in size to be transformed.