775 and . KT2440 7 DNA for -attgaattc 1 − (Fig. 1). The ∗ g ctcgtcgacaaa a µ Pp RI): 5 . Restriction sites P. putida Eco from -atcggtacc accC1phaC2 I): 5 entalScienceandForestry,Syracuse, gccacgtatctggtcagctt-3 lecular Chemistry, Graduate School Pp phaC2 ura, Department of Chemistry, State transformants 2009 Society of Chemical Industry .StrainsweregrowninLuria- Kpn 6 ( c and 10 , respectively. For determining PHA 1 ≥ − -atttctaga b P. putida ,andP1 phaC1 gggtt gatgatgctctggat-3 gmL I): 5 µ cluster deletion construct Xba pha and 20 -actaagctt 1 ;P2( − HI, and inserted into the same restriction site of pUC19, dIII): 5 gmL ) was removed from plasmid pGMR5 by restriction digest µ tember 2009 Published online in Wiley Interscience: 29 October 2009 Bam Hin and Christopher T. Nomura ( Division of Biotechnology andof Macromo Engineering, Hokkaido University, N13W8,060-8628, Kita-ku, Japan Sapporo-shi, Hokkaido Correspondence to: Christopher T. Nom UniversityofNewYork-CollegeofEnvironm NY 13210, USA. E-mail: [email protected] Department of Chemistry, State University of New York-College oftal Environmen- Science and Forestry, Syracuse, NY 13210, USA ∗ a b b accC1 ompetent cells. Furthermore, this transformation procedure yielding the plasmid, pUC19::phaC1 with content, the cells were grown on mineral salts (MS) media using various strains of Bertani (LB) medium (1%NaCl) tryptone, with the 0.5% appropriate antibiotic yeast when necessary. extract, For selection of and 0.5% transformants, kanamycinadded (Km) to and LB agar gentamycin platesof 50 and (Gm) liquid were media at final concentrations aggtgaacatggatgccttc-3 are underlined. Resultant fragmentsrestriction were sites of cloned pUC19. A into gene encoding the gentamycin( resistance same caaagcaa -3 P2 Construction of Homologous regions of were amplified byengineered PCR restriction with enzyme two sites, sets P1 of ( primers harboring ucing polyhydroxyalkanoate (PHA) biopolymers, a simple and ; polyhydroxyalkanoates (PHAs) in- DNA for replicative plasmids and Chean Ring Leong, 1 − a g cells for transformation via electroporation was developed. This method P. putida µ P.putida ,andonePHA cluster that in- competent cells JM109. The gene DNA. The transfor- pha phaC2 1 Pseudomonas putida Despite having a fully − Seiichi Taguchi g Pseudomonas and µ transformants b 1–3 7 10 KT2440, and linear DNA into Escherichiacoli phaC1 ≥ An easy, rapid, and inexpensive : 775–778 www.soci.org Escherichia coli 4–6 85 . The transformation efficiencies were transformants P. putida 7 2010; species were generated by transformation of linear DNA and these mutations were verified by PCR and the functions of many ORFs in this organ- strains to knock out the phaZ is a Gram-negative soil bacterium that plays Alexander P. Mueller, –10 3 4 a transformation; electroporation; Pseudomonas

Pseudomonas

2009 Society of Chemical Industry c depolymerase gene, within the range 10 way to efficiently transfer plasmidsby and electroporation linear of DNA freshly into platedprocedures cells can has be been done within developed. 10 All minused or for less. This transformation method of can be replicativeknockout mutant plasmids and site-specific or gene replacement. linear The transfor- DNAmation for procedure was confirmed by transferring severalthat plasmids can be replicatedseveral in cludes two PHA polymerase genes, sequenced genome, mation efficiency of cells prepared bywith that this of commercially method available is comparable and this method is adequate for many applications. linear DNA, which are comparablecan with be commercially performed available in c several less than 10 min, saving a great deal of time compared with traditional methods. Knockout mutants of analysis of PHA content. Keywords: replacement and plasmid transformation experiments were done J Chem Technol Biotechnol Strains, media, and growth conditions Table 1 shows the bacterial strains andPlasmids plasmids used in were this study. maintained in MATERIALS AND METHODS rapid method to prepare freshly plated can be usedefficiencies to were in transfer the range both of replicative plasmids and linear DNA to knock out genes into the cells. The transformation INTRODUCTION Pseudomonas putida animportantroleinelementcycling innature,bioremediation,and production of polyhydroxyalkanoates (PHAs), whichmentally friendly are biodegradable environ- plastics. Abstract In order to genetically modify microorganisms capable of prod bacteria Qin Wang, Ken’ichiro Matsumoto, transformation of biopolymer-producing (www.interscience.wiley.com) DOI 10.1002/jctb.2284 Quick and efficient method for genetic Technical Note Received: 29 July 2009 Revised: 14 September 2009 Accepted: 14 Sep clude conjugation, chemical transformation, and electroporation, which are all time consuming. ism remain unknown. A simplemethod and is required time to efficient functionally transformation analyze newlyThe identified traditional genes. methods of introducing new genes into . : 1 F; C, − ◦ µ et al and P3 : 775–778 for 10 min 85 8 g 10 14 15 16 L 15% sterile (40 s at 94 µ C overnight. ◦ This study × C, 250 rpm. After 2010; KCl, 20 mmol L ◦ 1 KT2440 at different − that amplify a region C, 30 Dr Niels-Ulrich Frigaard All experiments were knockout mutant ◦ C. 7 Cor37 ◦ ◦ pha M15] Takara P. putida 4). The cell suspensions were  . 0 15 mg) were treated with 2 mL of ∼ ∼ L of the transformation mixtures were C), 8 min at 72 600 µ ◦ J Chem Technol Biotechnol proAB lacIq Z [F L or 100 lac µ -caccaccatggacaaccaggttgctttgtttgtcg-3 − KT2440 and knockout mutant strains with a set of test λ Re C, 4 min at 72 ◦ Pp ,phaB ;withanECM399electroporationsystem(BTX,MA,USA) C and lyophilized for a minimum of 24 h. The cell dry weight C for 140 min. A total of 0.5 mL chloroform mixture containing Re P.putida  ◦ ◦ KT2442 12 supE44 relA1 -gctttcgaacttgctgtccagcaagctgaccagat-3 ) at various voltages. A0.5% volume of yeast 1 extract, mL 0.05% SOC NaCl, media (2% 2.5 mmol tryptone, L microcentrifuge tubes from the plates,sterile and resuspended 15% in glycerol 1 mL (OD thesolublemethylesterand0.5 mL 0.1%caprilicacidinchloroformwere mixed well, and these samples were assayedGas using a Chromatograph GC 2010 (Shimadzu, Japan). of (2612 bp for WTconditions and were one 1106 cycle bp for for 4 the min knockout). at The 94 reaction 5 was measured. The dry cells ( methanol-sulfuric acid (85 : 15)100 solution and 2 mL chloroform at incubation, 10 spread on to selective plates and incubated at 30 RESULTS AND DISCUSSION Transformation with replicative plasmids and linear DNA The broad hostthe transformation plasmid efficiencies pBBR1-MCS2 of was used to evaluate glucose) was added toThe cell each mixtures cuvette were then immediately transferredtubes after to and incubated sterile pulsing. for microcentrifuge 0 h or 1 h at 30 PCR amplification for verification of PCR reactions were done usingtype genomic DNA isolated from wild- primers, P3: 5 150 centrifuged at 13 000 rpmremoved. for The 1 min, cells andglycerol, were the supernatants and washed were then threeglycerol resuspended times for electroporation. again with Approximately 10 with 15% ng ofor 100 plasmid sterile linear DNA DNA was added tosuspensions the prepared were cells, and thecuvettes transferred DNA–cell (BTX, USA.) to on ice. 2 mm The cuvettes gap were pulsed electroporation at 36 GC analysis of PHA composition Liquid MS cultures (100 mL) were centrifugedat at 4 5000 performed in triplicate. 40 s at 60 ), phaC2 + ,phaA r K R Ps m − P. putida )Invitrogen K ,Km (r after (STQK) accC1 Plac , www.soci.org Q Wang r , accC1(Gen P. putida + Pp phaC1 M ,P2andp2 ,Amp into pUC19 to r P. putida hsd pp promoter ( 2009 Society of Chemical Industry were the primers R1 mutant of KT2440 9 cluster of c phaC1 r lac + hsd dIII to yield the linear derivative from KT2440 11 phaC2 in genome of wild-type pha ,the − r mt-2 ,mob Hin r ), and phaC2 ,Car R r ,RpoN ,Sm r r RI and (Gen ,and P. putida spontaneous rif recA1 endA1 gyrA96 thi-1 hsdR17 pUC19 derivative, phaC1 ,andP3andP3 Eco accC1 Pp , pp phaC1, phaZ KT2440 GPp104 PHA synthase negative mutant of IFO14164 Wild type rpoN Km were the primers to amplify phaC1 accC1phaC2 C overnight. Cells were transferred to 1.5 mL ◦ JM109 Pp sp. ATCC 29 347 OCT plasmid, PHA production 13 Bacterial strains and plasmids used in this study cells were streaked on LB agar plates and incubated Proposed process for knocking out the .P1andP1 Cluster including Cor37 ◦ pha Pseudomonas putida Pseudomonas putida Pseudomonas putida Pseudomonas putida Pseudomonas putida KT2442 Escherichia coli Table 1. Strain or plasmid Relevant characteristics Source or reference pGMR5 pUC19 derivative, Gm pJRD215pBBRSTQKAB pBBR1-MCS2 derivative, Km pUC19::phaC1 pBBR1-MCS2 Broad-host-range vector, lacPOZ pUC19 Amp Pseudomonas for verifying the knockout mutant.double crossover. D. Knockout mutant of KT2440. A. Insertion of www.interscience.wiley.com/jctb plasmid was then digested with were the primers to amplify phaC2 Figure 1. at 30 Preparation of competent cells and transformation by electroporation P. putida DNA for transformation. construct pUC19:C1GC2. B. The linear DNA digestedThe from pUC19:C1GC2. C. P. putida

776 777 6 6 6 5 5 1 2 5 . . pha 0 0 10 10 10 10 10 10 Pseu- ± ± × × × × × × 4 1 1 0 5 0 6 5 ...... The band 3 1 2 3 1 5 5 6 5 5 4 cluster and that 10 10 10 10 10 10 efficiencies P. putida × × × × × Transformation × 4 6 3 4 3 pha 5 . . . . . 46 49 . . 8 6 1 2 1 0 0 ± ± cluster and and the lower b 9 8 . . r r r r r r pha s of linear DNA of several roducts from chromosomal DNA crossover event to knockout the colonies, and the size of the PCR www.interscience.wiley.com/jctb 462 463 . . 0 0 P. putida Composition (mol %) ± ± 1 5 . . . 1 − Transformation efficiencie species KT2440IFO14164 2029 2029 bp bprpoN Gm GPp104 Gm KT2442 2029 2029 bp bp 2029 bp Gm Gm Gm KT2440 (14%), it was not entirely eliminated, indicating Verification of knockout mutations by PCR. Lanes 1 and 10 are mol L 3 128 224 − . . 0 0 10 . sp. ATCC 29 347 2029 bp Gm ± ± domonas Table 3. SpeciesP. putida P. putida P P. putida SizeP. putida P. putida Selection 0 h 1 h × 6 6 . . cluster. product is 2.6 kb.isolated Lanes from 4 gentamycin to resistant 9at transformants are of 2.6 PCR kb products isband for at the 1.1 chromosomal kb wild DNA represents theappeared type amplified to be knockout products copy mutation. for Lanes a 4 of double- and 9 the Figure 2. markers. Lanes 2isolated and from 3 wild-type have PCR p from which theon DNA MS had medium been tostill isolated determine produced in PHA about content. laneproduction 4% The 4 CDW was knockout was of significantly cells PHAP. grown putida lower (Table 4). than Although thatthat the PHA of strain may still the be merodiploid wild-type at the the wild type PCR amplicon was beyond(Fig. the 2). detection limit of PCR 35 . ), to 4 6 6 pp 4 10 10 10 C DNA ◦ 10 – – × × × 1 cluster. species 0 0 7 12 42 × − 2009 Society of Chemical Industry . . . . . phaC1 g 4 1 6 0 0 0 . c µ pha ± ± HD, 3-hydroxydecanoate; 3HDD, 3-hydroxydodecanoate. cluster. DNA 4 7 7 3 6 DNA without 0 0 . . 10 10 10 10 10 1 C37 efficiencies ◦ Cl concentration of 1 DNA without a − ) was transferred pha 4 × × × × × Transformation KT 2440 and its knockout mutant strain g pp 0 4 3 0 2 1 µ . . . . . KT2440, gentamycin − Pseudomonas g µ gene cluster with the phaC2 P. putida (a fragment of transformants ) PHA% 3HHx 3HO 3HD 3HDD pha 1 6 P.putida − 1200 V2000 1 V2500 2 V2500 5 V2500 1 V 1 02 13 01 4 . . 10 0 0 DNA, which might be due to the 0 h), which reduced the overall DNA with 1 h recovery time after (g L phaC1 : 775–778 1 × ± ± a 1 transformants r r r r r − = − 2 2 85 1 5 . . g . DNA.,Cellstransformedat2500 Vand g 0 µ T strains to knock out the 1 µ 10 transformants − (a fragment of g 6 × to 3 2010; cluster amplicon. The transformed strain µ 6 4 . –10 pha 10 4 phaC2 C had the highest transformation efficiency. The × ◦ r transformants Pseudomonas transformants KT2440 ), and PHA accumulation in wild-type Effect of various parameters on transformation efficiencies 4 R 6 Gen KT2440 0 10 transformants 10 7 × (Gen × 10 cluster, the other containing the knockout 2 P. putida . 3HHx, 3-hydroxyhexanoate; 3HO, 3-hydroxyoctanoate, 3 × CDW, cell dry weight. The linear DNA containing 3 P. putida pJRD215 10.2 kb Km All the cells werea grown on MS medium withb the NH P. putida Table 4. Strains CDW pBBR1-MCS2pBBR1-MCS2 5.1 kbpBBRSTQKAB 5.1 kb 8.8 Km kb Km Km DNApBBR1-MCS2 5.1 kb Size Km Selection Voltage 30 of Table 2. . Genetic transformation of biopolymer-producing bacteria www.soci.org The transformation efficiencies of different ranged from 9 knockout construct (Fig. 2). Thefrom results individually showed isolated the colonies DNA in isolated two lane 5, copies 6, of 7 and chromosomalpha 8 DNA, contained one containing theisolated wild-type from individually isolatedonly colonies the in knockout lane 4 and 9 had incubated at 30 recovery time. The transformation efficiency ofonly 1 pBBRSTQKAB was plasmid pJRD215 (10.2 kb) is another broad host rangeis plasmid but much larger than pBBR1-MCS2, hadof 1 a transformation efficiency into several J Chem Technol Biotechnol voltages, when the cells were incubated at(Table different 2). temperatures The transformation efficiencies ranged from 1 transformation and 8 (Table 3). This study alsocan showed that still transformation efficiencies reach 10 accC1 overexpression of PhaC1(STQK), PhaA, and PhaB proteins affecting the growth of the cells. 5 resistance colonies were selected and checkedthe by PCR replacement to confirm of the wild type Confirmation ofproduction knockout mutantAfter linear DNA was transferred with into PCR and PHA transformation time to less than 10 min. recovery incubation time ( . Appl Gene et al :574–577 : 775–778 :1104–1109 25 85 61 :275–280 (1987). :237–247 (1981). 2010; 51 16 Proc Natl Acad Sci USA Gene Gene sp. 61-3 from sugar. . Appl Microbiol . :4326–4333 (1989). y a cold-stable alkane-oxidizing tibiotic-resistance cassettes. oate copolymer production by 171 nks. V. pJRD215, a wide-host-range Appl Environ Microbiol :1457–1464 (2004). Pseudomonas Pseudomonas ) and polyhydroxyalkanoate synthesis 5 .Identificationandsequencesofgenesand :2191–2198 (1991). :363–370 (1996). fabH RpoN sigma factor in regulation of various strains. and cloning of genes for the entire regulated J Bacteriol 45 J Chem Technol Biotechnol 266 Pseudomonas oleovorans JBiolChem Biomacromolecules , Four new derivatives of the broad-host-range cloning vector , Effective enhancement of short-chain-length-medium- , Specific-purpose plasmid cloning vectors. II. Broad host range, :175–176 (1995). :7458–7462 (1981). Microbiol Biotechnol Molecular and functional analysis ofPseudomonas the putida TOL plasmid pWWO from aromatic ring meta78 cleavage pathway. 166 Vectors with restriction site ba cosmid vector with multiple cloning sites. coexpression ofprotein genetically synthase engineered IIIgenes. 3-ketoacyl-acyl-carrier- ( (1973). et al pBBR1MCS, carrying different an et al chain-length polyhydroxyalkan copolymer of 3-hydroxybutyric acidhydroxyalkanoaic and acids medium-chain-length-3- by sequencing ofapplication gyrB genes toPseudomonas putida with the universal(1995). primers detection and and their Pseudomonas taxonomic putida analysismetabolic functions. of Witholt B, MetabolismPseudomonasoleovorans of poly(3-hydroxyalkanoates)function of (PHAs) the encoded by proteins inof the PHA. synthesis and degradation et al high copy number,system RSF1010-derived for gene vectors, cloning in and a host-vector isolate of 8 Franklin FC, Bagdasarian M, Bagdasarian MM and Timmis KN, 7 Kato M, Bao HJ, Kang CK, Fukui T and Doi Y, Production of a novel 9 Bagdasarian M, Lurz R, Ruckert B, Franklin FC, Bagdasarian MM, Frey J, 13 Schwartz RD, Octene epoxidation b 15 Davison J, Heusterspreute M, Chevalier N, Ha-Thi V and Brunel F, 14 Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd 16 Nomura CT, Tanaka T, Gan Z, Kuwabara K, Abe H, Takase K, 10 Yamamoto S and Harayama S, PCR amplification and11 Kohler T, direct Harayama S, Ramos JL and12 Timmis KN, Huisman GW, Involvement Wonink of E, Meima R, Kazemier B, Terpstra P and U: www.soci.org Q Wang species. :3647–3651 Mol Microbiol Pseudomonas 72 2009 Society of Chemical Industry c with plasmid-linked DNA for replicative 1 Pseudomonas putida − Pseudomonas g µ DNA for linear DNA. This :38–44 (1988). 1 − :799–808 (2002). g 170 ,Completegenomesequenceand 4 easing microbial richness: missing µ Escherichia coli Principles of Gene Manipulation and et al Proc Natl Acad Sci USA and :649–657 (2001). 3 transformants 7 Anal Biochem 10 ≥ transformants Environ Microbiol 6 10 , 7th edn. Blackwell, Malden, MA and Oxford, UK ≥ KT2440. Environ Microbiol , Two different pathways are involved in the beta-oxidation of Pseudomonas putida :863–874 (2001). Bioaugmentation of soils by incr links. et al n-alkanoic and n-phenylalkanoic acidsgenetic in studies and39 biotechnological applications. comparative analysis of theputida metabolically versatile drug-resistance factor DNA. (1975). by electroporation. Genomics (2006). of 1 Dejonghe W, Boon N, Seghers D, Top EM and Verstraete W, 2 Olivera ER, Carnicero D, Garcia B, Minambres B, Moreno MA, Canedo L, 5 Fiedler S and Wirth R, Transformation of bacteria with plasmid DNA 3 Nelson KE,Weinel C,Paulsen IT, 6PrimroseSBandTwymanRM, 4 Chakrabarty AM, Mylroie JR, Friello DA and Vacca JG, Transformation plasmids and www.interscience.wiley.com/jctb REFERENCES All procedures can be done within 10ciencies min. could The transformation reach effi- The authors would like to thank DrpGMR5. Niels-Ulrik This work Frigaard has for been supported plasmid by a McIntire-Stennis Award and NSF DMR 0 907 085 to C.T. Nomura. ACKNOWLEDGEMENTS This paper describes a simple and rapid method totive transfer replica- plasmids and linear DNA into several CONCLUSIONS method can be usedand for expression transferring and entire linear plasmids DNA forreplacements. for knockout cloning mutations and gene

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