Transformation efficiency

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 that 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.[1] The number of viable cells in a preparation for a transformation reaction may range from 2x108 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 the smaller 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).[2]

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.

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 around 2-4x1010 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.[3] A number of factors may affect the transformation efficiency:[1]

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.[3]

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.[3] 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 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.[4]

Methods of transformation — 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, 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,[5] and inactivation of ligase or chloroform extration 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 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.[6] 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.

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.[7] 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.

See also

References

  1. 1 2 Hanahan, D.; Jessee, J.; Bloom, F. R. (1991). "Plasmid transformation of Escherichia coli and other bacteria". Methods in enzymology 204: 63–113. doi:10.1016/0076-6879(91)04006-a. PMID 1943786.
  2. ↑ "Calculating Transformation Efficiency". Sigma-Aldrich.
  3. 1 2 3 Hanahan D (1983). "Studies on the transformation of Escherichia coli with plasmids". J. Mol. Biol. 166 (4): 557–580. doi:10.1016/S0022-2836(83)80284-8. PMID 6345791.
  4. ↑ Tang, X.; Nakata, Y.; Li, H. O.; Zhang, M.; Gao, H.; Fujita, A.; Sakatsume, O.; Ohta, T.; Yokoyama, K. (1994). "The optimization of preparations of competent cells for transformation of E. Coli". Nucleic Acids Research 22 (14): 2857–2858. doi:10.1093/nar/22.14.2857. PMC 308259. PMID 8052542.
  5. ↑ Ymer, S. (1991). "Heat inactivation of DNA ligase prior to electroporation increases transformation efficiency". Nucleic Acids Research 19 (24): 6960. doi:10.1093/nar/19.24.6960. PMC 329344. PMID 1762931.
  6. ↑ Inoue, H.; Nojima, H.; Okayama, H. (1990). "High efficiency transformation of Escherichia coli with plasmids". Gene 96 (1): 23–28. doi:10.1016/0378-1119(90)90336-P. PMID 2265755.
  7. ↑ Gründemann D, Schömig E. (1996). "Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light" (PDF). Biotechniques 21 (5): 898–903. PMID 8922632.

External links

This article is issued from Wikipedia - version of the Sunday, April 03, 2016. The text is available under the Creative Commons Attribution/Share Alike but additional terms may apply for the media files.