A colony to-lawn method for efficient transformation of escherichia coli


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A colony to-lawn method for efficient transformation of escherichia coli

  1. 1. Letters in Applied Microbiology ISSN 0266-8254ORIGINAL ARTICLEA colony-to-lawn method for efficient transformation ofEscherichia coliY. An, A. Lv and W. WuInstitute of Applied Ecology, Chinese Academy of Sciences, Shenyang, ChinaKeywords Abstractchemical transformation, competent cells,electroporation, low-copy-number plasmid, Aims: To develop a fast, convenient, inexpensive and efficient Escherichia colimutant library. transformation method for changing hosts of plasmids, which can also facilitate the selection of positive clones after DNA ligation and transformation.Correspondence Methods and Results: A single fresh colony from plasmid-containing donorWenfang Wu, Institute of Applied Ecology, strain is picked up and suspended in 75% ethanol. Cells are pelleted and resus-Chinese Academy of Sciences. No.72 WenhuaRoad. Shenyang 110016, China. pended in CaCl2 solution and lysed by repetitive freeze–thaw cycles to obtainE-mail: wshr100@sina.com.cn plasmid-containing cell lysate. The E. coli recipient cells are scraped from the lawn of LB plate and directly suspended in the plasmid-containing cell lysate2010 ⁄ 0411: received 11 March 2010, revised for transformation. Additionally, a process based on colony-to-lawn transfor-4 April 2010 and accepted 21 April 2010 mation and protein expression was designed and conveniently used to screen positive clones after DNA ligation and transformation.doi:10.1111/j.1472-765X.2010.02864.x Conclusions: With this method, a single colony from plasmid-containing donor strain can be directly used to transform recipient cells scraped from lawn of LB plate. Additionally, in combination with this method, screening of positive clones after DNA ligation and transformation can be convenient and time-saving. Significance and Impact of the Study: Compared with current methods, this procedure saves the steps of plasmid extraction and competent cell preparation. Therefore, the method should be highly valuable especially for high-throughput changing hosts of plasmids during mutant library creation. efficiency (Okamoto et al. 1997; McCormac et al. 1998).Introduction In addition, a liposome-mediated transformation systemChanging hosts of plasmids by transformation is essential has been developed, because bacterial cells were found tofor many experiments in molecular biology, molecular be susceptible to transformation by liposomes (Kawatagenetics, etc. The CaCl2-mediated chemical transforma- et al. 2003). Although the methods described earlier havetion is one of the most commonly used transformation provided various choices for efficient transformation ofmethods until now. With this method, after treatment Escherichia coli, they are all dependent on the extractionwith CaCl2, a transient state of ‘competence’ is introduced of plasmid DNA beforehand. Therefore, when changingto the recipient cells, and the cells are more likely to the hosts of hundreds or thousands of plasmids isincorporate bacteriophage DNA or plasmid DNA (Man- performed, the work should be very time-consuming,del and Higa 1970; Cohen et al. 1972; Oishi and Cosloy expensive and inconvenient.1972). Some modified methods have been designed to In addition, during molecular cloning or constructionpromote the efficiency of chemical transformation (Golub of mutant libaries, frameshift mutations often occur,1988; Liu and Rashidbaigi 1990; Tang et al. 1994; Pope which may prevent the expression of proper proteins inand Kent 1996; Chen et al. 2001; Zeng et al. 2006). E. coli. Although these mutations can be detected andAnother efficient transformation method is electro- removed by DNA sequencing of randomly selected clones,poration, which can introduce a higher transformation the process is inconvenient especially when changing ª 2010 The Authors98 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103
  2. 2. Y. An et al. How to make transformation more efficienthosts of multiple plasmids are performed during muta- BL21(DE3) harbouring pETM11-P450-BM3 obtainedtion library creation. To address these problems, we either from colony-to-lawn transformation or from chem-describe a rapid, convenient and inexpensive method for ical transformation were cultured in TB media supple-changing E. coli hosts of plasmids. Additionally, based on mented with kanamycin. The cultures were induced usingthis method and protein expression, a process was IPTG (0Æ2 mmol l)1) at the exponential growth phase anddesigned and conveniently used to screen positive clones incubated at 20°C with shaking at 150 rev min)1 over-after DNA ligation and transformation. night. As a control, two colonies from the E. coli JM109 strain harbouring pETM11-P450-BM3 were also used for induced protein expression as described earlier. CellsMaterials and methods from these cultures were pelleted by centrifugation andEscherichia coli JM109 strains harbouring plasmids pUC19 checked the expression levels of protein P450-BM3 by(Ampr), pBR322 (Ampr), pYES2 (Ampr), pLysS (Camr), SDS-PAGE.pSE380 (Ampr), pETM-11 (Kanr) and pETM11-p450BM3 A mutant library of P450-BM3 was generated by error-(Kanr) were grown on antibiotic-supplemented LB agar prone PCR. The primers P450-For (5¢-GAGGGATACCA-plates for 36 h. The concentrations of the antibiotics TGGCAATTAAAGAAATGCCTCAGCC-3¢) and P450-Revampicillin, chloramphenicol and kanamycin were 50, 30 (5¢-CTCGCGGCCGCTTACCCAGCCCACACGTCTTTTG-and 50 mg l)1 respectively. For each strain, a single CG-3¢) were used for PCR amplification. The PCR wascolony was carefully picked up without gouging the agar. performed in mixture containing 2 ng of P450-BM3 tem-Each colony was suspended in a tube containing 200 ll plate DNA, 0Æ5 lmol l)1 both primers, 1 mmol l)1Milli-Q water followed by the addition of 600 ll ethanol d(C ⁄ T)TP, 0Æ2 mmol l)1 d(A ⁄ G)TP, 40 nmol l)1 MgCl2,to the tube. The mixtures were put in room temperature 1· Taq polymerase buffer and 3 Unit Taq polymerasefor 5 min, and then the cells were pelleted by centrifuga- with a total volume of 50 ll. This reaction mixture wastion. The tubes were put upside down for 10 min at heated at 95°C for 2 min followed by 30 cycles of incuba-room temperature to dry pellets, and a 30-ll aliquot of tion at 95°C for 1 min, 48°C for 40 s, and 72°C for0Æ1 mol l)1 CaCl2 was added to each tube and mixed 5 min and a final incubation at 72°C for 10 min. Aftercarefully. Then, the cells of different strains were lysed by purification, the PCR product was digested with NotI andfrozen at )80°C and thawed at 100°C for three cycles to NcoI and cloned into the corresponding restrictionobtain plasmid-containing cell lysates. The recipient strain enzyme sites of pETM11 vector and transformed intoBL21(DE3) was intensively grown on LB agar plates for E. coli JM109. Ten randomly selected transformants were24 h to form lawn. The cells from lawn were carefully used to transform E. coli recipient strain BL21(DE3) withscraped without gouging the agar and resuspended in five the colony-to-lawn transformation method. After trans-times volume of ice-cold water. A 30-ll aliquot of cells formation, transformed bacteria were grown in 50-mlsuspension was transferred to each tube containing the auto-inducing media (ZYM-5052) (Studier 2005). Theplasmid-containing cell lysate and mixed gently. The mix- cultures were first incubated at 37°C till OD600 = 1 andtures were incubated on ice for 15 min followed by heat then incubated at 20°C overnight with shaking atshock at 42°C for 40 s to perform transformation. Trans- 150 rev min)1. Cells from 5 ml of each culture were pel-formed bacteria were grown and selected by standard leted by centrifugation and used to check protein expres-methods. The number of transformants after each trans- sion by SDS-PAGE, and the remaining cultures (aboutformation with a single colony of plasmid-containing 45 ml for each) were kept at 4°C. The plasmids weredonor strain was calculated after incubation at 37°C for extracted from the remaining cultures of positive clones24 h. After each transformation, the plasmids were with expected protein expression, and DNA sequencingextracted from five randomly selected transformants and was performed.re-transformed into BL21(DE3) competent cells with thetraditional chemical transformation method. This was Resultsused to check whether the antibiotic-resistant colonieswere real transformants or just E. coli mutants or contam- The colony-to-lawn transformation method for changinginants. As a control, the cell lysates were directly spread hosts of plasmids is illustrated in Fig. 1a. The first step ison antibiotic-supplemented LB agar plates to check preparation of plasmid-containing cell lysate. A singlewhether all the cells were sterilized after 75% ethanol colony from plasmid donor strain is suspended in 75%incubation and freeze–thaw cycles. Changing hosts of ethanol followed by centrifugation to get pellet, and thenplasmid pETM11-P450-BM3 from JM109 to BL21(DE3) the cells are resuspended in CaCl2 solution and lysedwas also performed with chemical transformation after by freeze–thaw cycles to obtain plasmid-containing cellplasmid extraction. Then, two transformants of lysate. The second step is preparation of recipient cells forª 2010 The AuthorsJournal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 99
  3. 3. How to make transformation more efficient Y. An et al. (a) (b) Colonies from Colonies from Colonies from plasmid-containing plasmid-containing plasmid-containing Lawn from donor strain donor strain recipient strain donor strain Lawn from recipient strain A single colony Liquid culture 200 µl water Cells pelleted by Cells scraped 600 µl ethanol centrifugation from lawn Water Suspension Competent cell preparation 30 µl CaCI2 Freeze-thaw solution cycles Plasmid extraction Transformation Transformation Transformation Chemical transformation Colony-to-lawn transformation (c) (d) M 1 2 3 120 Transformation frequencies 100 120- 80 100- 85- 60 40 50- 20 (kDa) 0 pUC19 pBR322 pYES2 pLysS pSE380 pETM-11 pETM 11- p450BM3 PlasmidsFigure 1 The colony-to-lawn transformation method used for changing hosts of plasmid. (a) Outline of the colony-to-lawn transformationmethod. A single colony from plasmid donor strain is washed with 75% ethanol and air-dried, and then cells are suspended in CaCl2 solution andlysed by freeze–thaw cycles to obtain plasmid-containing cell lysate. At the same time, cells of plasmid recipient strain are scraped carefully fromfresh lawn and suspended in ice-cold water. Then, the recipient cells and plasmid-containing cell lysate are mixed gently and performed transfor-mation by heat shock method. The transformed bacteria are grown and selected by standard methods. (b) Comparison of the colony-to-lawntransformation method and the chemical transformation method. Plasmid extraction and competent cell preparation are essential steps for chemi-cal transformation, but not necessary for colony-to-lawn transformation. (c) SDS-PAGE gel shows protein expression of P450-BM3 before andafter changing hosts of pETM11-P450-BM3 either by colony-to-lawn transformation or by chemical transformation. Lane M: protein molecularweight marker; lane 1, after host changing of pETM11-P450-BM3 with the chemical transformation method; lanes 2, after host changing ofpETM11-P450-BM3 with the colony-to-lawn transformation method; lanes 3, before host changing of pETM11-P450-BM3 (i.e. protein expressedin Escherichia coli JM109). (d) The numbers of transformants obtained by changing hosts of various plasmids with the colony-to-lawn transforma-tion method. Each value represents the mean of five independent experiments.transformation. Cells of E. coli recipient strain are scraped plasmid extraction and competent cell preparation stepscarefully from fresh lawn without gouging the agar and are needed (Fig. 1b). Using pETM11-P450-BM3 as a sam-then suspended in ice-cold water. The third step is trans- ple, we changed its hosts from E. coli JM109 toformation. An aliquot of recipient cells and plasmid-con- BL21(DE3) either by colony-to-lawn transformation or bytaining cell lysate are mixed gently and we performed chemical transformation. After IPTG induction, the simi-transformation by heat shock method. Then, transformed lar expression levels of P450-BM3 protein were obtainedbacteria are grown and selected by standard methods. (Fig. 1c), indicating that there is no fundamental differ-The colony-to-lawn transformation method is more ence between these transformants. We tested the colony-convenient and rapid than current methods, because no to-lawn transformation method by using it to change the ª 2010 The Authors100 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103
  4. 4. Y. An et al. How to make transformation more efficienthosts of various plasmids, including the low-copy-number containing cell lysate, indicating that 75% ethanol incuba-plasmid pLysS. As a result, no less than 60 transformants tion and freeze–thaw cycles were efficient for sterilization,were available after each transformation with a single col- and no transformants obtained after transformation wereony of plasmid-containing donor strain (Fig. 1d). Addi- mutants or contaminants.tionally, the method is very convenient, because the LB A process based on colony-to-lawn transformation andagar plates with colonies of donor strains and recipient protein expression was designed and conveniently used tostrain can be stocked at 4°C for at least 7 days without remove frameshift mutations during the construction ofaffecting the transformation obviously (data not shown). mutant library (Fig. 2a). Recombinant plasmids areAs a control, plasmids from the randomly selected trans- constructed and transformed into E. coli cloning strain,formants were successfully re-transformed into E. coli followed by changing the hosts of plasmids from cloningBL21(DE3) by chemical transformation, indicating that strain to expression strain with the colony-to-lawn trans-the antibiotic-resistant colonies after colony-based trans- formation method. Then, randomly selected transfor-formation were real transformants but not E. coli mutants mants are cultured in auto-inducing media overnight. Anor contaminants. In addition, no colony was found on the aliquot of each culture is used to check protein expressionantibiotic-containing agar plates spread with the plasmid- by SDS-PAGE, and only the positive clones having (a) Construction of (b) Construction of recombinant plasmid recombinant plasmid Transformation Transformation Colonies of Colonies of plasmid-containing plasmid-containing donor strain donor strain Colony-based Overnight cultures transformation derived from One day single colonies 1 2 3 4 Liquid culture of Less than recipient cells two days 1 2 3 4 1 3 Extract plasmid from each clone 2 4 Transformation into 1 2 3 4 recipient cells 1 3 Induced expressionExtract plasmids from 1 2 3 4 positive clones SDS-Page analysis One day 1 2 3 4 DNA Sequencing or functional analysis SDS-Page analysis DNA Sequencing or functional analysisFigure 2 Protein expression in combination with the colony-to-lawn transformation method to screen in-frame clones from mutant library. (a)Outline of the experimental strategy. Plasmids from mutant library construction were changed hosts from cloning strain to expression strain withthe colony-to-lawn transformation method. Then, the randomly selected transformants are checked for protein expression by SDS-PAGE. Plasmidsare extracted for positive clones, and DNA sequencing or next round of mutagenesis was performed (shown as dotted line). (b) The chemicaltransformation method used for the same purpose. Plasmids are extracted from randomly selected clones after mutant library construction andtransformed into competent cells of expression strain for protein expression and SDS-PAGE analysis. The plasmids extracted from the clones whichhave expected protein expression are used for DNA sequencing or next round of mutagenesis (shown as dotted line).ª 2010 The AuthorsJournal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 101
  5. 5. How to make transformation more efficient Y. An et al.expected protein expression are used to extract plasmids incorrect protein expression in E. coli. Therefore, expres-from their remaining cultures, and DNA sequencing or sion of proteins (especially for the well expressedanother round of mutagenesis was performed Although proteins) can be used to predict whether the genes arethe current transformation methods can be used for the in-frame, which can be further determined by DNAsame purpose, the process should be less convenient, sequencing. This strategy is reasonable because lessbecause more time and an additional experimental step plasmids need to be extracted for DNA sequencing.(competent cell preparation) are needed (Fig. 2b). Addi- Therefore, a process based on colony-to-lawn transforma-tionally, more plasmids should to be extracted, because tion and protein expression provides a convenient way tothe clones used for plasmid extraction are before protein screen in-frame clones from mutant libraries.expression screening. In this work, the recombinant plas- In conclusion, as a simple and convenient DNA trans-mids with random mutations of P450-BM3 gene intro- formation strategy, this method may find wide applica-duced by error-prone PCR were used to test this method. tions in bioscience and biotechnology, especially whenThe recombinant plasmids were changed hosts from clon- changing hosts of multiple plasmids is needed.ing strain JM109 to expression strain BL21(DE3) with thecolony-to-lawn transformation method. Then, ten randomly Acknowledgementsselected transformants were used to check protein expres-sion levels, five of them were found to have expected The authors thank Sergi Castellano and Promdonkoyprotein expression. The plasmids were extracted and Patcharee for helpful discussions and review of this man-DNA sequencing was performed, and as a result, all the uscript.DNA sequences of positive clones were found to be in thecorrect open reading frames. References Chen, X., Guo, P., Xie, Z. and Shen, P. (2001) ADiscussion convenient and rapid method for genetic transformationWith this method, 75% ethanol is used for suspension of of E. coli with plasmids. Antonie Van Leeuwenhoek 80,the colony, because it has the functions of sterilization, 297–300.DNA sedimentation and pellet washing at the same time. Cohen, S.N., Chang, A.C.Y. and Hsu, L. (1972) Nonchromo-Therefore, this treatment can avoid contamination of the somal antibiotic resistance in bacteria: genetic transforma-plasmid donor strain after transformation and at the tion of Escherichia coli by R-factor DNA. Proc Natl Acadsame time reduce the loss of plasmid DNA during pellet Sci U S A 69, 2110–2114. Golub, E.I. (1988) ‘One minute’ transformation of competentwashing. It is worth noting that E. coli cells from colony E. coli by plasmid DNA. Nucleic Acids Res 16, 1641.are difficult to suspend directly in 75% ethanol, so the Kawata, Y., Yano, S. and Kojima, H. (2003) Escherichia colicells should be first suspended in water and then in 75% can be transformed by a liposome-mediated lipofectionethanol by adding proper volume of ethanol to the sus- method. Biosci Biotechnol Biochem 67, 1179–1181.pension. In addition, the recipient cells are conveniently Liu, H.Y. and Rashidbaigi, A. (1990) Comparison of variousprepared, and repeated washing and centrifugation steps competent cell preparation methods for high efficiencyfor preparing competent cells are not indispensable. This DNA transformation. BioTechniques 8, 21.is because the cells are grown on plate but not in liquid Mandel, M. and Higa, A. (1970) Calcium-dependent bacterio-culture, and there is no need to remove residual medium phage DNA infection. J Mol Biol 53, 159–162.from cell pellet by washing. Although only a small McCormac, A.C., Elliott, M.C. and Chen, D.F. (1998) A simplenumber of transformants can be obtained after colony-to- method for the production of highly competent cells oflawn transformation, in fact the number of transformants Agrobacterium for transformation via electroporation. Molis not a limiting factor for changing hosts of plasmids in Biotechnol 9, 155–159.most cases. It is because even thousands of transformants Oishi, M. and Cosloy, S.D. (1972) The genetic and biochemi-can be obtained after transformation, and only one of cal basis of the transformability of Escherichia coli K12.them is needed for the subsequent experiments. Because Biochem Biophys Res Commun 49, 1568–1572.of its simplicity and convenience, the method should be Okamoto, A., Kosugi, A., Koizumi, Y., Yanagida, F. andvaluable especially for high-throughput changing hosts of Udaka, S. (1997) High efficiency transformation of Bacillusplasmids during mutant library creation and functional brevis by electroporation. Biosci Biotechnol Biochem 61,analysis. 202–203. Frameshift mutations often occur during molecular Pope, B. and Kent, H.M. (1996) High efficiency 5 min trans-cloning or construction of mutant libraries. It is worth formation of Escherichia coli. 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  6. 6. Y. An et al. How to make transformation more efficientStudier, F.W. (2005) Protein production by auto-induction transformation of E. coli. Nucleic Acids Res 22, 2857– in high-density shaking cultures. Protein Expr Purif 41, 2858. 207–234. Zeng, W., Deng, Y., Yang, Z., Yuan, W., Huang, W., Zhu, C.,Tang, X., Nakata, Y., Li, H.O., Zhang, M., Gao, H., Bai, Y., Li, Y. et al. (2006) high transformation efficiency of Fujita, A., Sakatsume, O., Ohta, T. et al. (1994) The Escherichia coli with plasmids by adding amino modified optimization of preparations of competent cells for silica-nanoparticles. Biotechnology 5, 341–343.ª 2010 The AuthorsJournal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 103