of successful transformation events. 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, i.e. larger plasmids transform less well than smaller plasmids. 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. 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. 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. 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. 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. 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