Editing Genetic transformation (section)

==Practical aspects of transformation in molecular biology==
{{further|Transformation efficiency}}
The discovery of artificially induced competence in bacteria allow bacteria such as ''[[Escherichia coli]]'' to be used as a convenient host for the manipulation of DNA as well as expressing proteins.  Typically plasmids are used for transformation in ''E. coli''.  In order to be stably maintained in the cell, a plasmid DNA molecule must contain an [[origin of replication]], which allows it to be replicated in the cell independently of the replication of the cell's own chromosome.

The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as [[transformation efficiency]] and is measured in colony forming unit (cfu) per μg DNA used.  A transformation efficiency of 1×10<sup>8</sup> cfu/μg for a small plasmid like [[pUC19]] is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.

In [[calcium chloride transformation]], the cells are prepared by chilling cells in the presence of {{chem|Ca|2+}} (in {{chem|link=calcium chloride|CaCl|2}} solution), making the cell become permeable to [[plasmid|plasmid DNA]]. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42&nbsp;°C for 30–120 seconds).  This method works very well for circular plasmid DNA.  Non-commercial preparations should normally give 10<sup>6</sup> to 10<sup>7</sup> transformants per microgram of plasmid; a poor preparation will be about 10<sup>4</sup>/μg or less, but a good preparation of competent cells can give up to ~10<sup>8</sup> colonies per microgram of plasmid.<ref>[http://faculty.plattsburgh.edu/donald.slish/Transformation.html Bacterial Transformation] {{Webarchive|url=https://web.archive.org/web/20100610064730/http://faculty.plattsburgh.edu/donald.slish/Transformation.html |date=2010-06-10 }}</ref> Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 10<sup>9</sup>.<ref>{{cite journal | vauthors = Inoue H, Nojima H, Okayama H | title = High efficiency transformation of Escherichia coli with plasmids | journal = Gene | volume = 96 | issue = 1 | pages = 23–8 | date = November 1990 | pmid = 2265755 | doi = 10.1016/0378-1119(90)90336-P }}</ref> The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native [[exonuclease]] enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.
  
The transformation efficiency using the {{chem|CaCl|2}} method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA.<ref>{{cite journal|vauthors=Donahue RA, Bloom FR | title =Transformation efficiency of ''E. coli'' electroporated with large plasmid DNA |journal=Focus |volume=20 |issue=3 |pages=77–78 |date=September 1998 |url=http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.31180.File.dat/Focus%20Volume%2020%20Issue%203.pdf |url-status=unfit |archive-url=https://web.archive.org/web/20110903023521/http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.31180.File.dat/Focus%20Volume%2020%20Issue%203.pdf |archive-date=September 3, 2011 }}</ref> Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the electroporation process.

===Selection and screening in plasmid transformation===
Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid.<ref name="bimboim">{{cite journal | vauthors = Birnboim HC, Doly J | title = A rapid alkaline extraction procedure for screening recombinant plasmid DNA | journal = Nucleic Acids Research | volume = 7 | issue = 6 | pages = 1513–23 | date = November 1979 | pmid = 388356 | pmc = 342324 | doi = 10.1093/nar/7.6.1513 }}</ref> The plasmid therefore requires a [[selectable marker]] such that those cells without the plasmid may be killed or have their growth arrested. [[Antibiotic resistance]] is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain [[auxotrophy|auxotrophic]] markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow.

In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the insert. [[Reporter gene]]s can be used as [[marker gene|markers]], such as the ''[[lac operon|lacZ]]'' gene which codes for [[Beta-galactosidase|β-galactosidase]] used in [[blue-white screen]]ing. This method of screening relies on the principle of α-[[Complementation (genetics)|complementation]], where a fragment of the ''lacZ'' gene (''lacZα'') in the plasmid can complement another mutant ''lacZ'' gene (''lacZΔM15'') in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing ''lacZ-α'' is transformed into a ''lacZΔM15'' cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing [[X-gal]], forming characteristic blue colonies. However, the [[multiple cloning site]], where a gene of interest may be [[DNA ligase|ligated]] into the plasmid [[Vector (molecular biology)|vector]], is located within the ''lacZα'' gene. Successful ligation therefore disrupts the ''lacZα'' gene, and no functional β-galactosidase can form, resulting in white colonies.  Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.

Other commonly used reporter genes are [[green fluorescent protein]] (GFP), which produces cells that glow green under blue light, and the enzyme [[luciferase]], which catalyzes a reaction with [[luciferin]] to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.