Transformation Efficiency - Factors Affecting Transformation Efficiency

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 size. Individual cells are capable of taking up many DNA molecules, and that the presence of multiple plasmids does not significantly affect the occurrence of successful transformation events.

Forms of DNA — Supercoiled plasmid have a slightly 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 cellsE. 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 inactivation 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 1 x 109. Electroporation method in general has better transformation efficiency than chemical methods with over 1 x 1010 cfu/μg DNA possible, and it allows large plasmids of 200 kb in size to be transformed.

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