View source for Genetic transformation

{{Short description|Genetic alteration of a cell by uptake of genetic material from the environment}}
{{distinguish|text=an unrelated process called [[malignant transformation]] which occurs in the progression of [[cancer]]}}
[[File:Bacterial Transformation.svg|thumbnail|In this image, a gene from one bacterial cell is moved to another bacterial cell. This process of the second bacterial cell taking up new genetic material is called transformation.|422x422px]]

In [[molecular biology]] and [[molecular genetics|genetics]], '''transformation''' is the [[Introduction to genetics|genetic]] alteration of a [[cell (biology)|cell]] resulting from the direct uptake and incorporation of [[exogenous DNA|exogenous genetic material]] from its surroundings through the [[cell membrane]](s). For transformation to take place, the recipient bacterium must be in a state of [[Competence (biology)|competence]], which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.<ref name=Johnston/>

Transformation is one of three processes that lead to [[horizontal gene transfer]], in which exogenous genetic material passes from one bacterium to another, the other two being [[bacterial conjugation|conjugation]] (transfer of [[plasmid|genetic material]] between two bacterial cells in direct contact) and [[transduction (genetics)|transduction]] (injection of foreign DNA by a [[bacteriophage]] virus into the host bacterium).<ref name=Johnston>{{cite journal | vauthors = Johnston C, Martin B, Fichant G, Polard P, Claverys JP | title = Bacterial transformation: distribution, shared mechanisms and divergent control | journal = Nature Reviews. Microbiology | volume = 12 | issue = 3 | pages = 181–96 | date = March 2014 | pmid = 24509783 | doi = 10.1038/nrmicro3199 | s2cid = 23559881 }}</ref> In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.<ref name=Johnston/>

As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between [[Gram-positive bacteria|Gram-positive]] and [[Gram-negative bacteria]]; the number might be an overestimate since several of the reports are supported by single papers.<ref name=Johnston/>

"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "[[Malignant transformation|transformation]]" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "[[transfection]]".<ref name = "Alberts">{{cite book | title = Molecular Biology of the Cell | first1 = Bruce | last1 = Alberts | first2 = Alexander | last2 = Johnson | first3 = Julian | last3 = Lewis | first4 = Martin | last4 = Raff | first5 = Keith | last5 = Roberts | first6 = Peter | last6 = Walter | name-list-style = vanc | author-link1 = Bruce Alberts | year = 2002 | publisher = Garland Science | location= New York| isbn = 978-0-8153-4072-0 | page= G:35 | url = https://www.ncbi.nlm.nih.gov/books/NBK21052/ }}</ref>

==History==
Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist [[Frederick Griffith]].<ref name="Griffith_1928">{{cite journal | vauthors = Griffith F | title = The Significance of Pneumococcal Types | journal = The Journal of Hygiene | volume = 27 | issue = 2 | pages = 113–59 | year = 1928 | pmid = 20474956 | pmc = 2167760 | doi = 10.1017/s0022172400031879}}</ref> Griffith was interested in determining whether injections of heat-killed bacteria could be used to vaccinate mice against pneumonia.  However, he discovered that a non-virulent strain of ''[[Streptococcus pneumoniae]]'' could be made [[Virulence#Virulent bacteria|virulent]] after being exposed to heat-killed virulent strains. Griffith hypothesized that some "[[Griffith's experiment|transforming principle]]" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by [[Oswald Avery]], [[Colin Munro MacLeod|Colin MacLeod]], and [[Maclyn McCarty]]. They isolated DNA from a virulent strain of ''S. pneumoniae'' and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation" (See [[Avery-MacLeod-McCarty experiment]])<ref>Case, Christine; Funke, Berdell; Tortora, Gerard. Microbiology An Introduction(tenth edition)</ref> The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development of [[genetic markers]] and the discovery of other methods of genetic transfer ([[bacterial conjugation|conjugation]] in 1947 and [[Transduction (genetics)|transduction]] in 1953) by [[Joshua Lederberg]] that Avery's experiments were accepted.<ref>{{cite journal|title = The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in ''Anecdotal, Historical and Critical Commentaries on Genetics'' | journal = Genetics | volume = 136 | issue = 2 | pages = 423–6 | last = Lederberg | first = Joshua| author-link = Joshua Lederberg | year= 1994 | doi = 10.1093/genetics/136.2.423 | pmid = 8150273| pmc = 1205797 }}</ref>

It was originally thought that ''[[Escherichia coli]]'', a commonly used laboratory organism, was refractory to transformation.  However, in 1970, Morton Mandel and Akiko Higa showed that ''E. coli'' may be induced to take up DNA from [[Lambda phage|bacteriophage λ]] without the use of [[Helper virus|helper phage]] after treatment with calcium chloride solution.<ref>{{cite journal | vauthors = Mandel M, Higa A | title = Calcium-dependent bacteriophage DNA infection | journal = Journal of Molecular Biology | volume = 53 | issue = 1 | pages = 159–62 | date = October 1970 | pmid = 4922220 | doi = 10.1016/0022-2836(70)90051-3 }}</ref> Two years later in 1972, [[Stanley Norman Cohen]], Annie Chang and Leslie Hsu showed that {{chem|CaCl|2}} treatment is also effective for transformation of plasmid DNA.<ref>{{cite journal | vauthors = Cohen SN, Chang AC, Hsu L | title = Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 69 | issue = 8 | pages = 2110–4 | date = August 1972 | pmid = 4559594 | pmc = 426879 | doi = 10.1073/pnas.69.8.2110 | author-link1 = Stanley Norman Cohen | bibcode = 1972PNAS...69.2110C | doi-access = free }}</ref> The method of transformation by Mandel and Higa was later improved upon by [[Douglas Hanahan]].<ref>{{cite journal | vauthors = Hanahan D | title = Studies on transformation of Escherichia coli with plasmids | journal = Journal of Molecular Biology | volume = 166 | issue = 4 | pages = 557–80 | date = June 1983 | pmid = 6345791 | doi = 10.1016/S0022-2836(83)80284-8 | citeseerx = 10.1.1.460.2021 }}</ref> The discovery of artificially induced competence in ''E. coli'' created an efficient and convenient procedure for transforming bacteria which allows for simpler [[molecular cloning]] methods in [[biotechnology]] and [[research]], and it is now a routinely used laboratory procedure.

Transformation using [[electroporation]] was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of [[bacterial strain]]s that could be transformed.<ref>{{cite journal | vauthors = Wirth R, Friesenegger A, Fiedler S | title = Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation | journal = Molecular & General Genetics | volume = 216 | issue = 1 | pages = 175–7 | date = March 1989 | pmid = 2659971 | doi = 10.1007/BF00332248 | s2cid = 25214157 }}</ref> Transformation of animal and plant cells was also investigated with the first [[transgenic mouse]] being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982.<ref>{{cite journal | vauthors = Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM | title = Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes | journal = Nature | volume = 300 | issue = 5893 | pages = 611–5 | date = December 1982 | pmid = 6958982 | pmc = 4881848 | doi = 10.1038/300611a0 | bibcode = 1982Natur.300..611P }}</ref> In 1897 a bacterium that caused plant tumors, ''[[Agrobacterium tumefaciens]]'', was discovered and in the early 1970s the tumor-inducing agent was found to be a DNA [[plasmid]] called the [[Ti plasmid]].<ref>{{cite web |last = Nester| first = Eugene | url = http://www.apsnet.org/publications/apsnetfeatures/Pages/Agrobacterium.aspx|title = Agrobacterium: The Natural Genetic Engineer (100 Years Later) |website = APS|publisher = The American Phytopathological Society|access-date = 14 January 2011}}</ref> By removing the genes in the plasmid that caused the tumor and adding in novel genes, researchers were able to infect plants with ''A. tumefaciens'' and let the bacteria insert their chosen DNA into the genomes of the plants.<ref>{{cite journal | vauthors = Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J | title = Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity | journal = The EMBO Journal | volume = 2 | issue = 12 | pages = 2143–50 | year = 1983 | pmid = 16453482 | pmc = 555426 | doi = 10.1002/j.1460-2075.1983.tb01715.x }}</ref> Not all plant cells are susceptible to infection by ''A. tumefaciens'', so other methods were developed, including [[electroporation]] and [[Microinjection|micro-injection]].<ref>{{cite web |last = Peters| first = Pamela | url = http://www.accessexcellence.org/RC/AB/BA/Transforming_Plants.html|title = Transforming Plants - Basic Genetic Engineering Techniques|website = Access Excellence | access-date = 28 January 2010}}</ref> Particle bombardment was made possible with the invention of the [[Biolistic Particle Delivery System]] (gene gun) by [[John C. Sanford|John Sanford]] in the 1980s.<ref>{{cite news |title=Biologists invent gun for shooting cells with DNA |newspaper=Cornell Chronicle |page=3 |date=14 May 1987 |url=http://www.ecommons.cornell.edu/bitstream/1813/25239/1/018_33.pdf }}</ref><ref>{{cite journal  |vauthors=Sanford JC, Klein TM, Wolf ED, Allen N | title = Delivery of substances into cells and tissues using a particle bombardment process | journal = Journal of Particulate Science and Technology | volume = 5 | pages = 27–37 | year = 1987 | doi = 10.1080/02726358708904533 }}</ref><ref name="pmid1422046">{{cite journal | vauthors = Klein RM, Wolf ED, Wu R, Sanford JC | title = High-velocity microprojectiles for delivering nucleic acids into living cells. 1987 | journal = Biotechnology (Reading, Mass.) | volume = 24 | pages = 384–6 | year = 1992 | pmid = 1422046 }}</ref>

==Definitions==
Transformation is one of three forms of [[horizontal gene transfer]] that occur in nature among bacteria, in which DNA encoding for a trait passes from one bacterium to another and is integrated into the recipient genome by [[homologous recombination]]; the other two are [[Transduction (genetics)|transduction]], carried out by means of a [[bacteriophage]], and [[bacterial conjugation|conjugation]], in which a gene is passed through direct contact between bacteria.<ref name=Johnston/> In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.<ref name=Johnston/>

[[Competence (biology)|Competence]] refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory.<ref name=Johnston/>

It appears to be an ancient process inherited from a common prokaryotic ancestor that is a beneficial adaptation for promoting recombinational repair of DNA damage, especially damage acquired under stressful conditions. Natural genetic transformation appears to be an adaptation for repair of DNA damage that also generates [[genetic diversity]].<ref name=Johnston /><ref name=Michod />

Transformation has been studied in medically important [[Gram-negative bacteria]] species such as ''[[Helicobacter pylori]]'', ''[[Legionella pneumophila]]'', ''[[Neisseria meningitidis]]'', ''[[Neisseria gonorrhoeae]]'', ''[[Haemophilus influenzae]]'' and ''[[Vibrio cholerae]]''.<ref name=Seitz>{{cite journal | vauthors = Seitz P, Blokesch M | title = Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria | journal = FEMS Microbiology Reviews | volume = 37 | issue = 3 | pages = 336–63 | date = May 2013 | pmid = 22928673 | doi = 10.1111/j.1574-6976.2012.00353.x | url = https://infoscience.epfl.ch/record/181130/files/fmr353.pdf | doi-access = free }}</ref>  It has also been studied in Gram-negative species found in soil such as ''[[Pseudomonas stutzeri]]'', ''Acinetobacter baylyi'', and Gram-negative [[plant pathology|plant pathogens]] such as ''[[Ralstonia solanacearum]]'' and ''[[Xylella fastidiosa]]''.<ref name=Seitz /> Transformation among [[Gram-positive bacteria]] has been studied in medically important species such as ''[[Streptococcus pneumoniae]]'', ''[[Streptococcus mutans]]'', ''[[Staphylococcus aureus]]'' and ''[[Streptococcus sanguinis]]'' and in Gram-positive soil bacterium ''[[Bacillus subtilis]]''.<ref name=Michod>{{cite journal | vauthors = Michod RE, Bernstein H, Nedelcu AM | title = Adaptive value of sex in microbial pathogens | journal = Infection, Genetics and Evolution | volume = 8 | issue = 3 | pages = 267–85 | date = May 2008 | pmid = 18295550 | doi = 10.1016/j.meegid.2008.01.002 | bibcode = 2008InfGE...8..267M }}</ref> It has also been reported in at least 30 species of ''[[Pseudomonadota]]'' distributed in several different classes.<ref name="pmid17997281">{{cite journal | vauthors = Johnsborg O, Eldholm V, Håvarstein LS | title = Natural genetic transformation: prevalence, mechanisms and function | journal = Research in Microbiology | volume = 158 | issue = 10 | pages = 767–78 | date = December 2007 | pmid = 17997281 | doi = 10.1016/j.resmic.2007.09.004 | doi-access = free }}</ref> The best studied ''Pseudomonadota'' with respect to transformation are the medically important human pathogens ''[[Neisseria gonorrhoeae]]'', ''[[Haemophilus influenzae]]'', and ''[[Helicobacter pylori]]''.<ref name=Michod/>

"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "[[Malignant transformation|transformation]]" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "[[transfection]]".<ref name=Alberts/>

==Natural competence and transformation ==
{{main|Natural competence}}

Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).  The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of [[Pilus#Type IV pili|type IV pili]] and [[Secretion#Type II secretion system (T2SS)|type II secretion system]], as well as DNA [[translocase]] complex at the cytoplasmic membrane.<ref name=Chen />

Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the [[Cell membrane|cytoplasmic membrane]] via DNA translocase.<ref>{{cite journal | vauthors = Lacks S, Greenberg B, Neuberger M | title = Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 71 | issue = 6 | pages = 2305–9 | date = June 1974 | pmid = 4152205 | pmc = 388441 | doi = 10.1073/pnas.71.6.2305 | bibcode = 1974PNAS...71.2305L | doi-access = free }}</ref> Only single-stranded DNA may pass through, the other strand being degraded by nucleases in the process. The translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a [[RecA]]-dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. [[Pilin]] may be required for competence, but its role is uncertain.<ref>{{cite journal | vauthors = Long CD, Tobiason DM, Lazio MP, Kline KA, Seifert HS | title = Low-level pilin expression allows for substantial DNA transformation competence in Neisseria gonorrhoeae | journal = Infection and Immunity | volume = 71 | issue = 11 | pages = 6279–91 | date = November 2003 | pmid = 14573647 | pmc = 219589 | doi = 10.1128/iai.71.11.6279-6291.2003 }}</ref> The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.<ref>{{cite journal | vauthors = Sisco KL, Smith HO | title = Sequence-specific DNA uptake in Haemophilus transformation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 76 | issue = 2 | pages = 972–6 | date = February 1979 | pmid = 311478 | pmc = 383110 | doi = 10.1073/pnas.76.2.972 | bibcode = 1979PNAS...76..972S | doi-access = free }}</ref>

===Natural transformation===

Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be responsible for this process.<ref name=Chen>{{cite journal | vauthors = Chen I, Dubnau D | title = DNA uptake during bacterial transformation | journal = Nature Reviews. Microbiology | volume = 2 | issue = 3 | pages = 241–9 | date = March 2004 | pmid = 15083159 | doi = 10.1038/nrmicro844 | s2cid = 205499369 }}</ref><ref name="pmid17997281"/> In general, transformation is a complex, energy-requiring developmental process.  In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state. Competence development in ''[[Bacillus subtilis]]'' requires expression of about 40 genes.<ref>{{cite journal | vauthors = Solomon JM, Grossman AD | title = Who's competent and when: regulation of natural genetic competence in bacteria | journal = Trends in Genetics | volume = 12 | issue = 4 | pages = 150–5 | date = April 1996 | pmid = 8901420 | doi = 10.1016/0168-9525(96)10014-7 }}</ref> The DNA integrated into the host chromosome is usually (but with rare exceptions) derived from another bacterium of the same species, and is thus homologous to the resident chromosome.

In ''B. subtilis'' the length of the transferred DNA is greater than 1271 kb (more than 1 million bases).<ref>{{cite journal | vauthors = Saito Y, Taguchi H, Akamatsu T | title = Fate of transforming bacterial genome following incorporation into competent cells of Bacillus subtilis: a continuous length of incorporated DNA | journal = Journal of Bioscience and Bioengineering | volume = 101 | issue = 3 | pages = 257–62 | date = March 2006 | pmid = 16716928 | doi = 10.1263/jbb.101.257 }}</ref> The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb.<ref>{{cite journal | vauthors = Saito Y, Taguchi H, Akamatsu T | title = DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA | journal = Journal of Bioscience and Bioengineering | volume = 101 | issue = 4 | pages = 334–9 | date = April 2006 | pmid = 16716942 | doi = 10.1263/jbb.101.334 }}</ref> It appears that about 7-9% of the recipient cells take up an entire chromosome.<ref>{{cite journal | vauthors = Akamatsu T, Taguchi H | title = Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis | journal = Bioscience, Biotechnology, and Biochemistry | volume = 65 | issue = 4 | pages = 823–9 | date = April 2001 | pmid = 11388459 | doi = 10.1271/bbb.65.823 | s2cid = 30118947 | doi-access = free }}</ref>

The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla) are known to undergo this process.<ref name="pmid17997281"/>

Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth.  Transformation in ''[[Haemophilus influenzae]]'' occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase.<ref>{{cite journal | vauthors = Goodgal SH, Herriott RM | title = Studies on transformations of Hemophilus influenzae. I. Competence | journal = The Journal of General Physiology | volume = 44 | issue = 6 | pages = 1201–27 | date = July 1961 | pmid = 13707010 | pmc = 2195138 | doi = 10.1085/jgp.44.6.1201 }}</ref> Transformation in ''[[Streptococcus mutans]]'', as well as in many other streptococci, occurs at high cell density and is associated with [[biofilm]] formation.<ref>{{cite journal | vauthors = Aspiras MB, Ellen RP, Cvitkovitch DG | title = ComX activity of Streptococcus mutans growing in biofilms | journal = FEMS Microbiology Letters | volume = 238 | issue = 1 | pages = 167–74 | date = September 2004 | pmid = 15336418 | doi = 10.1016/j.femsle.2004.07.032 }}</ref> Competence in ''B. subtilis'' is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation.<ref>{{cite journal | vauthors = Anagnostopoulos C, Spizizen J | journal = Journal of Bacteriology | volume = 81 | issue = 5 | pages = 741–6 | date = May 1961 | pmid = 16561900 | pmc = 279084 | title = Requirements for Transformation in Bacillus Subtilis | doi = 10.1128/JB.81.5.741-746.1961 }}</ref> Similarly, in ''[[Micrococcus luteus]]'' (a representative of the less well studied ''[[Actinomycetota]]'' phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation.<ref>{{cite journal |last1=Angelov |first1=Angel |last2=Bergen |first2=Paul |last3=Nadler |first3=Florian |last4=Hornburg |first4=Philipp |last5=Lichev |first5=Antoni |last6=Ãœbelacker |first6=Maria |last7=Pachl |first7=Fiona |last8=Kuster |first8=Bernhard |last9=Liebl |first9=Wolfgang |title=Novel Flp pilus biogenesis-dependent natural transformation |journal=Frontiers in Microbiology |date=10 February 2015 |volume=6 |pages=84 |doi=10.3389/fmicb.2015.00084|pmid=25713572 |pmc=4322843 |doi-access=free }}</ref><ref>{{cite journal |last1=Lichev |first1=Antoni |last2=Angelov |first2=Angel |last3=Cucurull |first3=Inigo |last4=Liebl |first4=Wolfgang |title=Amino acids as nutritional factors and (p)ppGpp as an alarmone of the stringent response regulate natural transformation in Micrococcus luteus |journal=Scientific Reports |date=30 July 2019 |volume=9 |issue=1 |pages=11030 |doi=10.1038/s41598-019-47423-x | pmid = 31363120 | pmc = 6667448|bibcode=2019NatSR...911030L }}</ref>

By releasing intact host and plasmid DNA, certain [[bacteriophage]]s are thought to contribute to transformation.<ref name="KeenBliskovsky2017">{{cite journal | vauthors = Keen EC, Bliskovsky VV, Malagon F, Baker JD, Prince JS, Klaus JS, Adhya SL | title = Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation | journal = mBio | volume = 8 | issue = 1 | pages = e02115–16 | date = January 2017 | pmid = 28096488 | doi = 10.1128/mBio.02115-16 | pmc=5241400}}</ref>

===Transformation, as an adaptation for DNA repair===

Competence is specifically induced by DNA damaging conditions.  For instance, transformation is induced in ''Streptococcus pneumoniae'' by the DNA damaging agents mitomycin C (a DNA cross-linking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks).<ref>{{cite journal | vauthors = Claverys JP, Prudhomme M, Martin B | title = Induction of competence regulons as a general response to stress in gram-positive bacteria | journal = Annual Review of Microbiology | volume = 60 | pages = 451–75 | year = 2006 | pmid = 16771651 | doi = 10.1146/annurev.micro.60.080805.142139 }}</ref>  In ''B. subtilis'', transformation is increased by UV light, a DNA damaging agent.<ref>{{cite journal | vauthors = Michod RE, Wojciechowski MF, Hoelzer MA | title = DNA repair and the evolution of transformation in the bacterium Bacillus subtilis | journal = Genetics | volume = 118 | issue = 1 | pages = 31–9 | date = January 1988 | doi = 10.1093/genetics/118.1.31 | pmid = 8608929 | pmc = 1203263 | url = http://www.genetics.org/cgi/pmidlookup?view=long&pmid=8608929 }}</ref> In ''Helicobacter pylori'', ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation<ref>{{cite journal | vauthors = Dorer MS, Fero J, Salama NR | title = DNA damage triggers genetic exchange in Helicobacter pylori | journal = PLOS Pathogens | volume = 6 | issue = 7 | pages = e1001026 | date = July 2010 | pmid = 20686662 | pmc = 2912397 | doi = 10.1371/journal.ppat.1001026 | editor1-last = Blanke | editor1-first = Steven R | doi-access = free }}</ref> Using ''Legionella pneumophila'', Charpentier et al.<ref name=Charpentier>{{cite journal | vauthors = Charpentier X, Kay E, Schneider D, Shuman HA | title = Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila | journal = Journal of Bacteriology | volume = 193 | issue = 5 | pages = 1114–21 | date = March 2011 | pmid = 21169481 | pmc = 3067580 | doi = 10.1128/JB.01146-10 }}</ref> tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents, caused strong induction. These DNA damaging agents were mitomycin C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks<ref>{{cite journal | vauthors = Albertini S, Chételat AA, Miller B, Muster W, Pujadas E, Strobel R, Gocke E | title = Genotoxicity of 17 gyrase- and four mammalian topoisomerase II-poisons in prokaryotic and eukaryotic test systems | journal = Mutagenesis | volume = 10 | issue = 4 | pages = 343–51 | date = July 1995 | pmid = 7476271 | doi = 10.1093/mutage/10.4.343 }}</ref>), bicyclomycin (causes single- and double-strand breaks<ref>{{cite journal | vauthors = Washburn RS, Gottesman ME | title = Transcription termination maintains chromosome integrity | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 2 | pages = 792–7 | date = January 2011 | pmid = 21183718 | pmc = 3021005 | doi = 10.1073/pnas.1009564108 | bibcode = 2011PNAS..108..792W | doi-access = free }}</ref>), and hydroxyurea (induces DNA base oxidation<ref>{{cite journal | vauthors = Sakano K, Oikawa S, Hasegawa K, Kawanishi S | title = Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide | journal = Japanese Journal of Cancer Research | volume = 92 | issue = 11 | pages = 1166–74 | date = November 2001 | pmid = 11714440 | pmc = 5926660 | doi = 10.1111/j.1349-7006.2001.tb02136.x }}</ref>). UV light also induced competence in ''L. pneumophila''. Charpentier et al.<ref name=Charpentier /> suggested that competence for transformation probably evolved as a DNA damage response.  Natural transformation in the extraordinarily radiation resistant bacterium ''[[Deinococcus radiodurans]]'' is associated with the [[DNA repair|repair of DNA damage]] under stressful conditions.<ref>{{cite journal |vauthors=Sharma DK, Soni I, Rajpurohit YS |title=Surviving the storm: exploring the role of natural transformation in nutrition and DNA repair of stressed Deinococcus radiodurans |journal=Appl Environ Microbiol |volume=91 |issue=1 |pages=e0137124 |date=January 2025 |pmid=39651863 |pmc=11784314 |doi=10.1128/aem.01371-24 |url=}}</ref>
 
Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important [[DNA repair]] process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair process that is especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation.<ref name=Bernstein>{{cite book| vauthors = Bernstein H, Bernstein C, Michod RE | year = 2012 | chapter = Chapter 1: DNA repair as the primary adaptive function of sex in bacteria and eukaryotes | title = DNA Repair: New Research | veditors = Kimura S, Shimizu S | publisher =  Nova Sci. Publ., Hauppauge, N.Y. | pages = 1–49 | isbn = 978-1-62100-808-8 | chapter-url =https://www.novapublishers.com/catalog/product_info.php?products_id=31918 }}</ref>
 
To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using ''B. subtilis'' irradiated by UV light as the damaging agent (reviewed by Michod et al.<ref>{{cite journal | vauthors = Michod RE, Bernstein H, Nedelcu AM | title = Adaptive value of sex in microbial pathogens | journal = Infection, Genetics and Evolution | volume = 8 | issue = 3 | pages = 267–85 | date = May 2008 | pmid = 18295550 | doi = 10.1016/j.meegid.2008.01.002 | bibcode = 2008InfGE...8..267M | url = http://www.hummingbirds.arizona.edu/Faculty/Michod/Downloads/IGE%20review%20sex.pdf }}</ref> and Bernstein et al.<ref name=Bernstein />) The results of these experiments indicated that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular process responsible for repair was likely HRR.  Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes (see [[Evolution of sexual reproduction]]; [[Meiosis]].)

==Methods and mechanisms of transformation in laboratory ==
[[File:Artificial Bacterial Transformation.svg|thumb|391x391px|Schematic of bacterial transformation – for which artificial competence must first be induced]]

===Bacterial===
Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.<ref>{{cite periodical |vauthors=Donahue RA, Bloom FR |title=Large-volume transformation with high-throughput efficiency chemically competent cells |periodical=Focus |volume=20 |issue=2 |pages=54–56 |date=July 1998 |url=http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.78703.File.dat/Focus%20Volume%2020%20Issue%202.pdf |archive-url=https://web.archive.org/web/20130306120401/http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/focus.Par.78703.File.dat/Focus%20Volume%2020%20Issue%202.pdf |archive-date=2013-03-06 |oclc=12352630 |via=Invitrogen}}{{rs|date=September 2022|reason=corporate publication where the authors work for the corporation;}}</ref> Typically the cells are incubated in a solution containing [[divalent]] [[cation]]s (often [[calcium chloride]]) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are called competent cells.

It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to-[[lipopolysaccharide]] bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation.<ref name="Srivastava">{{cite book | last = Srivastava | first = Sheela | name-list-style = vanc | title = Genetics of Bacteria | publisher = [[Springer-Verlag]] | year = 2013 | location = India | url = https://link.springer.com/content/pdf/10.1007%2F978-81-322-1090-0.pdf#page=112| doi = 10.1007/978-81-322-1090-0 | isbn = 978-81-322-1089-4 | s2cid = 35917467 }}</ref> The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed – perhaps because of improved DNA accessibility.

The surface of bacteria such as ''E. coli'' is negatively charged due to [[phospholipids]] and [[lipopolysaccharide]]s on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.

DNA entry into ''E. coli'' cells is through channels known as zones of adhesion or Bayer's junction, with a typical cell carrying as many as 400 such zones. Their role was established when [[cobalamine]] (which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.<ref name="Srivastava" />

It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA.  The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall.

[[Electroporation]] is another method of promoting competence.  In this method the cells are briefly shocked with an [[electric field]] of 10-20 [[Volt|kV]]/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter.  After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms.

===Yeast===
Most species of [[yeast]], including ''[[Saccharomyces cerevisiae]]'', may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab.<ref>{{cite journal | vauthors = Kawai S, Hashimoto W, Murata K | title = Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism | journal = Bioengineered Bugs | volume = 1 | issue = 6 | pages = 395–403 | date = 1 November 2010 | pmid = 21468206 | doi = 10.4161/bbug.1.6.13257 | pmc=3056089}}</ref>
* Yeast cells may be treated with enzymes to degrade their cell walls, yielding [[spheroplast]]s. These cells are very fragile but take up foreign DNA at a high rate.<ref>{{cite journal | vauthors = Hinnen A, Hicks JB, Fink GR | title = Transformation of yeast | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 75 | issue = 4 | pages = 1929–33 | date = April 1978 | pmid = 347451 | pmc = 392455 | doi = 10.1073/pnas.75.4.1929 | bibcode = 1978PNAS...75.1929H | doi-access = free }}</ref>
* Exposing intact yeast cells to [[alkali]] [[cation]]s such as those of [[caesium]] or [[lithium]] allows the cells to take up plasmid DNA.<ref>{{cite journal | vauthors = Ito H, Fukuda Y, Murata K, Kimura A | title = Transformation of intact yeast cells treated with alkali cations | journal = Journal of Bacteriology | volume = 153 | issue = 1 | pages = 163–8 | date = January 1983 | doi = 10.1128/JB.153.1.163-168.1983 | pmid = 6336730 | pmc = 217353 }}</ref> Later protocols adapted this transformation method, using [[lithium acetate]], [[polyethylene glycol]], and single-stranded DNA.<ref>{{cite book | vauthors = Gietz RD, Woods RA | chapter = Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method | volume = 350 | pages = 87–96 | year = 2002 | pmid = 12073338 | doi = 10.1016/S0076-6879(02)50957-5 | isbn = 9780121822538 | series = Methods in Enzymology | title = Guide to Yeast Genetics and Molecular and Cell Biology - Part B }}</ref> In these protocols, the single-stranded DNA preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving it available for transformation.<ref>{{cite journal | vauthors = Gietz RD, Schiestl RH, Willems AR, Woods RA | title = Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure | journal = Yeast | volume = 11 | issue = 4 | pages = 355–60 | date = April 1995 | pmid = 7785336 | doi = 10.1002/yea.320110408 | s2cid = 22611810 }}</ref>
* [[Electroporation]]: Formation of transient holes in the cell membranes using electric shock; this allows DNA to enter as described above for bacteria.<ref name=":0">{{cite journal|last=Schiestl|first=Robert H.|author2=Manivasakam, P. |author3=Woods, Robin A. |author4= Gietzt, R.Daniel |title=Introducing DNA into Yeast by Transformation|journal=Methods|date=1 August 1993|volume=5|issue=2|pages=79–85|doi=10.1006/meth.1993.1011}}</ref>
* Enzymatic digestion<ref>{{cite journal|last=Spencer|first=F.|author2=Ketner, G. |author3=Connelly, C. |author4= Hieter, P. |title=Targeted Recombination-Based Cloning and Manipulation of Large DNA Segments in Yeast|journal=Methods|date=1 August 1993|volume=5|issue=2|pages=161–175|doi=10.1006/meth.1993.1021}}</ref> or agitation with glass beads<ref>{{cite journal | vauthors = Costanzo MC, Fox TD | title = Transformation of yeast by agitation with glass beads | journal = Genetics | volume = 120 | issue = 3 | pages = 667–70 | date = November 1988 | doi = 10.1093/genetics/120.3.667 | pmid = 3066683 | pmc = 1203545 }}</ref> may also be used to transform yeast cells.

'''Efficiency''' &ndash; Different yeast genera and species take up foreign DNA with different efficiencies.<ref name="Dohmen1991">{{cite journal | vauthors = Dohmen RJ, Strasser AW, Höner CB, Hollenberg CP | title = An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera | journal = Yeast | volume = 7 | issue = 7 | pages = 691–2 | date = October 1991 | pmid = 1776359 | doi = 10.1002/yea.320070704 | s2cid = 7108750 }}</ref> Also, most transformation protocols have been developed for baker's yeast, ''S. cerevisiae'', and thus may not be optimal for other species. Even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. cerevisiae strains were transformed with 10&nbsp;ug of plasmid YEp13, the strain DKD-5D-H yielded between 550 and 3115 colonies while strain OS1 yielded fewer than five colonies.<ref name="Hayama2002">{{cite journal | vauthors = Hayama Y, Fukuda Y, Kawai S, Hashimoto W, Murata K | title = Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells | journal = Journal of Bioscience and Bioengineering | volume = 94 | issue = 2 | pages = 166–71 | year = 2002 | pmid = 16233287 | doi=10.1016/s1389-1723(02)80138-4}}</ref>

===Plants===
<!-- This section is linked from [[Arabidopsis thaliana]] -->
A number of methods are available to transfer DNA into plant cells. Some [[vector (molecular biology)|vector]]-mediated methods are:
*''[[Agrobacterium]]''-mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for ten minutes in a fluid containing suspended ''Agrobacterium''. The bacteria will attach to many of the plant cells exposed by the cut. The plant cells secrete wound-related phenolic compounds which in turn act to upregulate the virulence operon of the Agrobacterium. The virulence operon includes many genes that encode for proteins that are part of a Type IV secretion system that exports from the bacterium proteins and DNA (delineated by specific recognition motifs called border sequences and excised as a single strand from the virulence plasmid) into the plant cell through a structure called a pilus. The transferred DNA (called T-DNA) is piloted to the plant cell nucleus by nuclear localization signals present in the Agrobacterium protein VirD2, which is covalently attached to the end of the T-DNA at the Right border (RB). Exactly how the T-DNA is integrated into the host plant genomic DNA is an active area of plant biology research. Assuming that a selection marker (such as an antibiotic resistance gene) was included in the T-DNA, the transformed plant tissue can be cultured on selective media to produce shoots. The shoots are then transferred to a different medium to promote root formation. Once roots begin to grow from the transgenic shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). The seeds from this first plant (called the T1, for first transgenic generation) can be planted on a selective (containing an antibiotic), or if an [[herbicide resistance]] gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants. Some plants species, such as ''Arabidopsis thaliana'' can be transformed by dipping the flowers or whole plant, into a suspension of ''Agrobacterium tumefaciens'', typically strain C58 (C=Cherry, 58=1958, the year in which this particular strain of ''A. tumefaciens'' was isolated from a cherry tree in an orchard at Cornell University in Ithaca, New York). Though many plants remain recalcitrant to transformation by this method, research is ongoing that continues to add to the list the species that have been successfully modified in this manner.
*[[Viral transformation]] ([[transduction (genetics)|transduction]]): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However, genomes of most plant viruses consist of single stranded [[RNA]] which replicates in the cytoplasm of infected cell. For such genomes this method is a form of [[transfection]] and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus-free and also free of the inserted gene.

Some vector-less methods include:
*[[Gene gun]]: Also referred to as particle bombardment, microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The [[transformation efficiency]] is lower than in ''Agrobacterium''-mediated transformation, but most plants can be transformed with this method.
*[[Electroporation]]: Formation of transient holes in cell membranes using electric pulses of high field strength; this allows DNA to enter as described above for bacteria.<ref name="ISC BIOLOGY">{{cite book | title=ISC BIOLOGY | publisher=Nageen Prakashan | author=V.Singh and D.K.Jain | chapter=Applications of recombinant DNA | year=2014 | pages=840}}</ref>

=== Fungi ===
There are some methods to produce transgenic [[Fungus|fungi]] most of them being analogous to those used for plants. However, fungi have to be treated differently due to some of their microscopic and biochemical traits:

* A major issue is the [[Dikaryon|dikaryotic state]] that parts of some fungi are in; dikaryotic cells contain two haploid nuclei, one of each parent fungus. If only one of these gets transformed, which is the rule, the percentage of transformed nuclei decreases after each [[Spore|sporulation]].<ref name=":1">{{Cite journal|last1=Poyedinok|first1=N. L.|last2=Blume|first2=Ya. B.|date=March 2018|title=Advances, Problems, and Prospects of Genetic Transformation of Fungi|journal=Cytology and Genetics|volume=52|issue=2|pages=139–154|doi=10.3103/S009545271802007X|s2cid=4561837|issn=0095-4527}}</ref>
* Fungal cell walls are quite thick hindering DNA uptake so (partial) removal is often required;<ref>{{Cite journal|last1=He|first1=Liya|last2=Feng|first2=Jiao|last3=Lu|first3=Sha|last4=Chen|first4=Zhiwen|last5=Chen|first5=Chunmei|last6=He|first6=Ya|last7=Yi|first7=Xiuwen|last8=Xi|first8=Liyan|date=2017|title=Genetic transformation of fungi|journal=The International Journal of Developmental Biology|volume=61|issue=6–7|pages=375–381|doi=10.1387/ijdb.160026lh|pmid=27528043|issn=0214-6282|doi-access=free}}</ref> complete degradation, which is sometimes necessary,<ref name=":1" /> yields [[protoplast]]s.
* Mycelial fungi consist of filamentous [[hypha]]e, which are, if at all, separated by internal cell walls interrupted by pores big enough to enable nutrients and organelles, sometimes even nuclei, to travel through each hypha. As a result, individual cells usually cannot be separated. This is problematic as neighbouring transformed cells may render untransformed ones immune to selection treatments, e.g. by delivering nutrients or proteins for antibiotic resistance.<ref name=":1" />
* Additionally, growth (and thereby mitosis) of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues.<ref name=":1" />

As stated earlier, an array of methods used for plant transformation do also work in fungi:

* Agrobacterium is not only capable of infecting plants but also fungi, however, unlike plants, fungi do not secrete the phenolic compounds necessary to trigger Agrobacterium so that they have to be added, e.g. in the form of [[acetosyringone]].<ref name=":1" />
* Thanks to development of an expression system for small RNAs in fungi the introduction of a [[CRISPR/Cas9-mediated genome editing|CRISPR/CAS9-system]] in fungal cells became possible.<ref name=":1" /> In 2016 the USDA declared that it will not regulate a white button mushroom strain edited with CRISPR/CAS9 to prevent fruit body browning causing a broad discussion about placing CRISPR/CAS9-edited crops on the market.<ref>{{Cite journal|last=Waltz|first=Emily|date=April 2016|title=Gene-edited CRISPR mushroom escapes US regulation|journal=Nature|volume=532|issue=7599|pages=293|doi=10.1038/nature.2016.19754|pmid=27111611|issn=0028-0836|bibcode=2016Natur.532..293W|doi-access=free}}</ref>
* Physical methods like electroporation, biolistics ("gene gun"), [[sonoporation]] that uses cavitation of gas bubbles produced by ultrasound to penetrate the cell membrane, etc. are also applicable to fungi.<ref>{{Cite journal|last1=Rivera|first1=Ana Leonor|last2=Magaña-Ortíz|first2=Denis|last3=Gómez-Lim|first3=Miguel|last4=Fernández|first4=Francisco|last5=Loske|first5=Achim M.|date=June 2014|title=Physical methods for genetic transformation of fungi and yeast|journal=Physics of Life Reviews|volume=11|issue=2|pages=184–203|doi=10.1016/j.plrev.2014.01.007|pmid=24507729|bibcode=2014PhLRv..11..184R}}</ref>

===Animals===
Introduction of DNA into animal cells is usually called [[transfection]], and is discussed in the corresponding article.

==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.

== References ==
{{reflist|33em}}

== External links ==
* [http://www.1lecture.com/Microbiology/Bacterial%20Transformation/index.html Bacterial Transformation ] (a Flash Animation)
* [https://web.archive.org/web/20110721174250/http://www.gmo-safety.eu/basic-info/602.ready-aim-fire.html "Ready, aim, fire!"] At the Max Planck Institute for Molecular Plant Physiology in Potsdam-Golm plant cells are 'bombarded' using a particle gun

{{Genetic recombination}}
{{Genetic engineering}}
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[[Category:Gene delivery]]
[[Category:Modification of genetic information]]
[[Category:Molecular biology]]