406:
recognize exogenous DNA based on differences in methylation patterns. To address this problem, strategies such as altering the methylation of the exogenous DNA using commercial methylases or reducing the restriction activity in the recipient cells have been applied. For example, using methylation-negative mutants or temporarily inactivating the restriction system with heat can reduce the recipient cell's ability to impose restrictions on the exogenous DNA.
274:– 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 its transformation efficiencies.
396:
to treat the cells. However, these methods resulted in transformation efficiencies, with a maximum of 10 - 10 colony forming units (CFU) per microgram of plasmid DNA. Later research found that certain cations, such as Mn, Ca, Ba, Sr and Mg could have a positive effect on transformation efficiencies,
306:
extraction 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 competent cells can yield transformation efficiency ranging from 10 to 10 cfu/μg DNA. Protocols for chemical method however exist
267:
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
172:
or other similarly sized or smaller vectors, such as the 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
132:
will have a specific gene or sequence that is not present in the host cell genome, and therefore can be used as a target for qPCR. By quantifying the number of copies of this specific gene or sequence in the transformed cells, it is possible to determine the amount of plasmid DNA present in the cell,
325:
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
284:
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
57:
and host cells. This efficiency can be affected by a number of factors, including the method used for introducing the DNA, the type of cell and plasmid used, and the conditions under which the transformation is performed. Therefore, measuring and optimizing transformation efficiency is an important
405:
Some bacterial cells have restriction-modification systems that can degrade exogenous plasmids that are foreign to the host cell. This can greatly reduce the efficiency of transformation. This is due to restriction systems in the recipient cells that target and destroy exogenous DNA. These systems
223:
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 10 lower
374:
larger than 30 kb, it is suggested to use electrocompetent cells that have transformation efficiencies of over 1 x 10 CFU/μg. This will ensure a high success rate in introducing the DNA and forming a large number of colonies. It is important to adjust and optimize the electroporation buffer
298:, temperature of heat shock, incubation time after heat shock, growth medium used, pH 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
78:
By measuring the transformation efficiency, we can utilize the information from our experiment to evaluate how effectively our transformation went. This is a quantification of how many cells were altered by 1 μg of plasmid DNA. In essence, it is a sign that the
330:
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
188:, the theoretical limit of transformation efficiency for most commonly used plasmids would be over 1×10 cfu/μg. In practice the best achievable result may be around 2–4×10 cfu/μg for a small plasmid like pUC19, and considerably lower for large plasmids.
285:
copy number origin of replication will generally have a higher transfection efficiency than one with a low copy number origin, using a plasmid with an origin of replication that is active in the host cell can lead to a higher transfection efficiency.
196:
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. A number of factors may affect the transformation efficiency:
244:
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
375:(Increasing the concentration of the electroporation buffer can result in increased transformation efficiencies ) and the shape, strength, number, and number of pulses these electrical parameters play a key role in transformation efficiency.
387:
can be performed in a simple laboratory setup, typically yielding transformation efficiencies that are adequate for cloning and subcloning applications, approximately 10 CFU/μg. One of the early methods used was a combination of
1354:
121:
Alternatively, CFUs can be reported per microgram of DNA used for the transformation. This can be calculated by multiplying the number of colonies by the volume of the culture plated and dividing by the amount of DNA used.
357:
Electroporation has been found to have an average yield typically between 10 - 10 CFU/ug . However, a transformation efficiencies as high as 0.5-5 x 10 colony forming units (CFU) per microgram of DNA for
176:
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.
1316:
Rhee MS, Kim JW, Qian Y, Ingram LO, Shanmugam KT (July 2007). "Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans".
595:
Rasala BA, Barrera DJ, Ng J, Plucinak TM, Rosenberg JN, Weeks DP, et al. (May 2013). "Expanding the spectral palette of fluorescent proteins for the green microalga
Chlamydomonas reinhardtii".
42:) per microgram of DNA added to the cells. A higher transformation efficiency means that more cells are able to take up the DNA, and a lower efficiency means that fewer cells are able to do so.
114:
To calculate the transformation efficiency, divide the number of colonies by the number of cells plated and multiply by 100. The result will be the transformation efficiency as a percentage.
354:
tends to be more efficient than chemical methods and can be applied to a wide range of species and to strains that were previously resistant and recalcitrant to transformation techniques.
49:, transformation efficiency is a crucial parameter, it is used to evaluate the ability of different methods to introduce plasmid DNA into cells and to compare the efficiency of different
230:– 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.
630:
1533:"Decreased electroporation efficiency in Borrelia burgdorferi containing linear plasmids lp25 and lp56: impact on transformation of infectious B. burgdorferi"
268:
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.
86:
Transformation efficiency is typically measured as the number of transformed cells per total number of cells. It can be represented as a percentage or as
1234:
Aune TE, Aachmann FL (February 2010). "Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed".
139:- This method relies on the use of a plasmid that contains a fluorescent protein or reporter gene. The transformed cells are then analyzed by
111:
Count the number of colonies that grow on the plates. This represents the number of cells that have taken up and expressed the plasmid DNA.
125:
737:
Nakata Y, Tang X, Yokoyama KK (1996). "Preparation of competent cells for high-efficiency plasmid transformation of
Escherichia coli".
1582:"Acquisition of maltose chemotaxis in Salmonella typhimurium by the introduction of the Escherichia coli chemosensory transducer gene"
136:
503:
Sieuwerts S, de Bok FA, Mols E, de vos WM, Vlieg JE (October 2008). "A simple and fast method for determining colony forming units".
754:
479:
317:
procedure for as little as 45 seconds can damage the DNA, and this can significantly reduce the transformation efficiency. Adding
1035:"Dissecting the effects of antibiotics on horizontal gene transfer: Analysis suggests a critical role of selection dynamics"
154:
The number of viable cells in a preparation for a transformation reaction may range from 2×10 to 10; most common methods of
118:
For example, if you plate 1x 10 cells and count 1000 colonies, the transformation efficiency is: (1000/1x 10) x 100 = 0.1%
108:
Incubate the plates for a period of time (usually overnight) at the appropriate temperature and conditions for the cells.
332:
314:
38:. The efficiency of transformation is typically measured as the number of transformants (cells that have taken up the
548:"Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv. Hezuo 908) with improved efficiency"
151:. The transformation efficiency is then calculated as the percentage of cells that express the fluorescent protein.
462:
Hanahan D, Jessee J, Bloom FR (1991). "[4] Plasmid transformation of
Escherichia coli and other bacteria".
97:
forming assay. Here is an example of how to calculate transformation efficiency using colony forming units (CFUs):
1155:
Inoue H, Nojima H, Okayama H (November 1990). "High efficiency transformation of
Escherichia coli with plasmids".
415:
384:
295:
80:
35:
1278:
236:– Cloning strains may contain mutations that improve the transformation efficiency of the cells. For example,
1192:"Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light"
1428:"Table 1: The Single Nucleotide Polymorphisms in cathepsin B protein mined from literature (PMID: 16492714)"
144:
54:
1445:
281:
1485:
986:"Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila"
839:"Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli"
435:
343:
The method used for introducing the DNA have a significant impact on the transformation efficiency.
148:
94:
87:
59:
184:
is roughly equivalent to 1 in 2000 molecules of the plasmid used being introduced into cells. In
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1108:"Heat inactivation of DNA ligase prior to electroporation increases transformation efficiency"
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46:
1279:"Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation"
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for making super competent cells that may yield a transformation efficiency of over 1 x 10.
164:
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741:. Methods in Molecular Biology. Vol. 69. New Jersey: Humana Press. pp. 129–137.
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Hanahan D (June 1983). "Studies on transformation of
Escherichia coli with plasmids".
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67:
39:
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One of the most common ways to measure transformation efficiency is by performing a
83:
experiment was successful. It should be determined under conditions of cell excess.
888:"The optimization of preparations of competent cells for transformation of E. coli"
63:
1631:"Increasing DNA transfer efficiency by temporary inactivation of host restriction"
1597:
1329:
952:
564:
547:
30:
refers to the ability of a cell to take up and incorporate exogenous DNA, such as
1436:
1427:
1277:
Romero D, Pérez-García A, Veening JW, de
Vicente A, Kuipers OP (September 2006).
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746:
690:"Transformation of Escherichia coli with large DNA molecules by electroporation"
367:
1478:
Proceedings of the
National Academy of Sciences of the United States of America
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303:
102:
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903:
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937:"Protoplast transformation of glutamate-producing bacteria with plasmid DNA"
886:
Tang X, Nakata Y, Li HO, Zhang M, Gao H, Fujita A, et al. (July 1994).
322:
17:
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1380:"High efficiency transformation of E. coli by high voltage electroporation"
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Sun S, Kang XP, Xing XJ, Xu XY, Cheng J, Zheng SW, Xing GM (2015-09-03).
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mutation can significantly improve the efficiency of its transformation.
1001:
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210:
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found that transformation efficiency declines linearly with increasing
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129:
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294:– The method of preparation of competent cells, the length of time of
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size, i.e. larger plasmids transform less well than smaller plasmids.
169:
180:
A transformation efficiency of 1×10 cfu/μg for a small plasmid like
158:
preparation yield around 10 viable cells per reaction. The standard
181:
1531:
Lawrenz MB, Kawabata H, Purser JE, Norris SJ (September 2002).
778:
Yan L, Xu R, Zhou Y, Gong Y, Dai S, Liu H, Bian Y (June 2019).
464:
Plasmid transformation of
Escherichia coli and other bacteria
984:
Charpentier X, Kay E, Schneider D, Shuman HA (March 2011).
313:– Exposure of DNA to UV radiation in standard preparative
780:"Effects of Medium Composition and Genetic Background on
466:. Methods in Enzymology. Vol. 204. pp. 63–113.
1355:"Competent Cell Selection–6 General Considerations - US"
162:
used for determination of transformation efficiency in
58:
step in many molecular biology applications, including
1580:
Mizuno T, Mutoh N, Panasenko SM, Imae Y (March 1986).
326:
wavelength UV produces weaker fluorescence with the
1349:
1347:
935:Katsumata R, Ozaki A, Oka T, Furuya A (July 1984).
401:
Restriction barriers to an efficient transformation
147:to determine the number of cells that express the
1474:"Genetic transformation in Escherichia coli K12"
1033:Lopatkin AJ, Sysoeva TA, You L (December 2016).
457:
455:
128:(qPCR) - This method utilizes the fact that the
1378:Dower WJ, Miller JF, Ragsdale CW (July 1988).
552:Biotechnology & Biotechnological Equipment
1674:Bacteria Transformation Efficiency Calculator
8:
436:"how to calculate transformation efficiency"
362:. For samples that are hard to handle, like
192:Factors affecting transformation efficiency
90:forming units (CFUs) per microgram of DNA.
1629:Edwards RA, Helm RA, Maloy SR (May 1999).
688:Sheng Y, Mancino V, Birren B (June 1995).
1646:
1605:
1556:
1507:
1497:
1435:
1403:
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1190:Gründemann D, Schömig E (November 1996).
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862:
813:
803:
713:
648:
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133:and thus the transformation efficiency.
784:-Mediated Transformation Efficiency of
631:"Calculating Transformation Efficiency"
427:
105:containing the appropriate antibiotics.
1454:
1443:
1236:Applied Microbiology and Biotechnology
224:efficiency than double-stranded ones.
397:with Mn showing the greatest effect.
7:
339:Efficiency of transformation methods
1472:Cosloy SD, Oishi M (January 1973).
1283:Journal of Microbiological Methods
25:
101:Plate a known number of cells on
1083:"Plasmids - an overview !Topics"
517:10.1111/j.1472-765X.2008.02417.x
302:, and inactivation of ligase or
173:is reached at over 10 ng).
1549:10.1128/IAI.70.9.4798-4804.2002
855:10.1128/JB.180.8.2063-2071.1998
505:Letters in Applied Microbiology
1:
1641:(5): 892–4, 896, 898 passim.
1598:10.1128/jb.165.3.890-895.1986
1330:10.1016/j.plasmid.2006.11.006
953:10.1128/jb.159.1.306-311.1984
667:10.1016/S0022-2836(83)80284-8
565:10.1080/13102818.2015.1056753
278:Plasmid origin of replication
1169:10.1016/0378-1119(90)90336-P
655:Journal of Molecular Biology
472:10.1016/0076-6879(91)04006-a
1295:10.1016/j.mimet.2006.01.005
383:Chemical transformation or
315:agarose gel electrophoresis
1705:
1437:10.7717/peerj.7425/table-1
34:, during a process called
1248:10.1007/s00253-009-2349-1
747:10.1385/0-89603-383-x:129
416:Transformation (genetics)
28:Transformation efficiency
1106:Ymer S (December 1991).
837:Murphy KC (April 1998).
1586:Journal of Bacteriology
990:Journal of Bacteriology
941:Journal of Bacteriology
843:Journal of Bacteriology
379:Chemical transformation
272:Presence of antibiotics
145:fluorescence microscopy
1537:Infection and Immunity
1453:Cite journal requires
1396:10.1093/nar/16.13.6127
1384:Nucleic Acids Research
1124:10.1093/nar/19.24.6960
1112:Nucleic Acids Research
1051:10.1002/bies.201600133
904:10.1093/nar/22.14.2857
892:Nucleic Acids Research
739:cDNA Library Protocols
706:10.1093/nar/23.11.1990
694:Nucleic Acids Research
1087:www.sciencedirect.com
805:10.3390/genes10060467
282:origin of replication
240:K12 strains with the
1499:10.1073/pnas.70.1.84
1359:www.thermofisher.com
335:and transformation.
1490:1973PNAS...70...84C
1002:10.1128/JB.01146-10
149:fluorescent protein
60:genetic engineering
261:Culture conditions
203:– A study done in
1648:10.2144/99265st02
1390:(13): 6127–6145.
1209:10.2144/96215rr02
1045:(12): 1283–1292.
898:(14): 2857–2858.
700:(11): 1990–1996.
609:10.1111/tpj.12165
597:The Plant Journal
234:Genotype of cells
228:Media composition
137:Fluorescent assay
47:molecular biology
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