IUBMB

Life, 00(00): 000–000, Month 2012

Research Communication
Fluorescent Protein Engineering by In Vivo Site-directed Mutagenesis
Melvys Valledor1,2, Qinghua Hu3, Paul Schiller1,2,4, and Richard S. Myers1
1

Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA
Geriatric Research, Education, and Clinical Center, and Research Service, Veteran’s Affairs Medical Center, Miami, FL, USA
3
Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA
4
Department of Orthopedics, University of Miami Miller School of Medicine, Miami, FL, USA
2

Summary
In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua,
Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo sitedirected mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. Ó 2012 IUBMB
IUBMB Life, 00: 000–000, 2012
Keywords

recombineering; fluorescent proteins; gene targeting; mutagenesis; recombination; oligonucleotides.

Abbreviations

CMV, cytomegalovirus; DMEM, Dulbecco’s modified
Eagle’s medium; E. coli, Escherichia coli; FBS, fetal bovine serum; FP, fluorescent protein; LB, lysogeny broth;
MIAMI, marrow-isolated adult multilineage inducible;
MMR, DNA mismatch repair; oligo, oligonucleotide.

ogy. Although in vitro mutagenesis has been used to modify the spectra of green FP and many spectral variants are commercially available, we have developed a simple method for producing spectrally distinct variants on demand by making modest changes to FP genes without subcloning. Based on previous studies (1–3) and our analysis of sequences encoding green, cyan, blue, and yellow FP variants (Supporting Information Fig.
1), amino acids at positions 64 (F|L), 65 (G|T), 66 (Y|W|H), and 203 (S|T|Y) appear to be primarily responsible for the
‘‘color’’ of each protein, whereas other residues are less likely to be important for spectral shifts. We surmised that it might be possible to produce several spectrally distinct FPs by oligonucleotide (oligo)-directed mutagenesis in vivo by recombineering.
Recombineering uses viral recombination proteins [e.g., Beta protein expressed from a defective lambda prophage (4, 5)] to anneal a mutagenic oligo to its homologous target sequence inside the cell (6). Unlike in vitro site-directed mutagenesis, recombineering can directly modify any size of DNA molecule, including chromosomes and large bacterial artificial chromosomes (7–9). As oligo-mediated recombineering is most efficient in bacteria, we manipulated gfp gene variants stably integrated into the Escherichia coli genome (10) or in lentiviral plasmid vectors (11) in E. coli.

EXPERIMENTAL PROCEDURES
INTRODUCTION
The ability to easily change the spectra of fluorescent proteins (FPs) expands the palette for routine molecular cell biolAdditional Supporting Information may be found in the online version of this article.
Received 2 March 2012; accepted 30 March 2012
Address correspondence to: Richard S. Myers, Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA. Tel: 1305-243-2056. Fax:
1305-243-3955. E-mail: r.myers@miami.edu
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.1041

Cell Culture
293T cells (obtained from Dr. Priya Rai) were grown in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose medium with 10% fetal bovine serum (FBS) and antibiotics–antimycotics (50 U/mL Penicillin G, 0.25 lg/mL Amphotericin B, and
10 lg/mL Streptomycin) at 37 8C in 5% atmospheric CO2. Marrow-isolated adult multilineage inducible (MIAMI) 3515 adult human stem cells (12) (isolated from whole bone marrow from a
20-year-old living male donor) were grown in DMEM-low glucose, 3% FBS, 20 mM ascorbic acid, 12.9 nM arachidonic acid,

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1.12 lM cholesterol, 290 nM DL-alpha-tocopherol acetate, 85.9 nM myristic acid, 69.4 nM oleic acid, 76.5 nM palmitic acid,
77.1 nM palmitoleic acid, 68.9 nM stearic acid, 100 U/mL penicillin, and 0.1 mg/mL streptomycin under low oxygen conditions
(3% O2, 5% CO2, and 92% N2) at 37 8C. Media were changed every 2–3 days and the cells were detached using trypsin upon reaching $ 60% confluency and pelleted. Pelleted cells were resuspended in medium and plated in 10 ng/mL fibronectintreated flasks at 100 cells per square centimeter.

Plasmids pDual-eGFP was created by ligating a PCR product [produced using 50 phosphorylated primers (oligos 61 and 62) to amplify the T7 promoter region from pET28a] to SmaI digested pNL-eGFP/CEF (11) and screening transformed RosettagamiTM2(DE3) colonies for gain of green fluorescence. The inserted sequence contains the T7 promoter, the LacI-binding site (Lac operator), a Shine–Dalgarno sequence, a His6 tag, and a T7 tag. The resulting His6–T7 tag–eGFP fusion protein is strongly expressed from pDual-eGFP in Rosetta-gamiTM2(DE3), and the construct was verified by sequencing using oligo 44 as a primer. pDual-eGFP(W66) and pDual-eGFP(Stop66) were created from pDual-eGFP by recombineering with oligos 59 and
60, respectively, in strain RIK473. pDual-eGFP(H66) and pDual-eGFP(Y66) were created from pDual-eGFP using the
QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent
Technologies, Santa Clara, CA, USA) with oligos 68–71. Mutagenesis was confirmed by allele-specific PCR (Y66 with oligos
49 and 45 or oligos 72 and 46, W66 with oligos 50 and 45,
Stop66 with oligos 53 and 45, H66 with oligos 73 and 46, T203 with oligos 45 and 74, and Y203 with oligos 45 and 75) and sequencing using oligos 45 and 46. pCMV-VSV-G and pCMVdR8.2dvpr used to create lentivirus-transducing particles were gifts from Dr. Priya Rai. All oligo sequences are listed in Supporting Information Table 2.
Recombineering
Recombineering was performed essentially as in ref. 4. Oligos were designed with the desired change centrally located with ‡35 nt of targeting homology at each side. Oligos were obtained from Sigma Genosys (St. Louis, MO, USA), polyacrylamide gel electrophoresis purified, resuspended in TE buffer (10 mM Tris and 1 mM EDTA, pH 7.55) to 100 ng/lL, and stored at 220 8C until used. All oligo sequences are listed in Supporting Information Table 2. Recombineering host strains were grown overnight in LB medium at 32 8C, diluted 1/100 into fresh LB, and grown at 32 8C to mid-log phase. Recombinase expression was induced at 42 8C for 15 min. Cells were chilled on ice for 10 min to stop induction and to prepare cells for electroporation. Cells were then washed in ice-cold water and concentrated 200-fold. Forty microliters of induced competent cells was mixed with 1 lL of oligo (100 ng); for plasmid recombineering, 10 ng of the plasmid containing the target gene

(13) was also added. Cells were electroporated in chilled 1-mm gap electroporation cuvettes using a Bio-Rad Gene Pulser (Hercules, CA, USA) at 1.75 kV, 200 O, and 25 lF. Cells were allowed to recover in 1 mL LB for 30 min at 32 8C before plating dilutions on LB agar medium and incubation at 32 8C. For
FP engineering in pDual-eGFP by recombineering, cultures were grown overnight in LB broth and then plasmid DNA was isolated and used to transform the E. coli Rosetta-gamiTM2 strain to permit expression of FP genes from the T7 promoter.
In all cases, fluorescent recombinant colonies were scored using a plate imager (DR46B Dark Reader transilluminator, Clare
Chemical Research, Dolores, CO, USA). Occasionally, recombinants were screened by allele-specific PCR. Recombination efficiency was calculated as the recombinant titer divided by the total titer. Transformation efficiency was determined by parallel electroporation of the recombineering strain with pUC19. Mutagenesis was validated by allele-specific PCR followed by sequencing. The detailed recombineering protocol is available in the Supporting Information Methods.

Confocal Microscopy
E. coli grown to OD600 5 0.4 in LB at 32 8C were pelleted and resuspended in 1.05% K2HPO4, 0.45% KH2PO4, 0.005%
MgSO4Á7H2O, 0.1% (NH4)2SO4, 0.05% sodium citrate, and 0.2% glucose to 10% of the initial volume. One microliter of the concentrated cells was added to slides with mounting medium
(P7481 Prolong Antifade, Molecular Probes, Eugene, OR, USA).
Slides were covered and left to sit overnight before imaging.
Images were collected with a Zeiss LSM710 confocal microscope
(Thornwood, NY, USA). PerkinElmer/Improvision Velocity 64bit software (Waltham, MA, USA) was used to create a maximum projection, and Adobe Photoshop CS3 (San Jose, CA,
USA) was used to merge the colors. MIAMI adult human stem cells were grown on fibronectin-coated cover slips. Image acquisition was performed with a Leica SP5 confocal microscope
(Mannheim, Germany) using a 633 oil-immersion 1.3 numerical aperture objective. Confocal acquisition parameters were determined at the beginning of the study, and the same parameters
(e.g., gains, slit aperture, and laser intensity) were used for all the images. Confocal optical sections were 0.6 lm thick. Field selection was performed using the 40 , 6-diamidino-2-phenylindole
(DAPI) channel to identify MIAMI cells. Adobe Photoshop (San
Jose, CA, USA) was used to merge the colors.
Fluorometry
Protein extracts were prepared from overnight E. coli cultures that were subsequently pelleted, incubated with BugBuster and Benzonase, and clarified by centrifugation as recommended by Novagen (Madison, WI, USA). Excitation and emission spectra were determined using a PTI QuantumMaster spectrofluorometer (Birmingham, NJ, USA) with 2-nm slits. The average integrated 1-sec samples detected $ 104–106 emitted photons. The data were collected using Felix (PTI, Birmingham,

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NJ, USA) and analyzed using Excel (Microsoft, Redmond, WA,
USA) and PRISM (GraphPad, La Jolla, CA, USA). Each spectrum was corrected for background, normalized to its peak value, and plotted.

Lentiviral Transduction
A total of 2 3 106 293T cells were plated on 10 cm2 dishes.
The next day, 4 lg of pDual-eGFP (or variants), 4 lg of pHR0 8.2DR, and 0.4 lg of pCMV-VSV-G were mixed with 24 lL of Fugene 6 (Roche, Indianapolis, IN, USA) and 400 lL of
DMEM, incubated for 15–30 min, and then added to the 293T cells. Cells were incubated overnight in a BL21 incubator. The next day, the medium was changed to MIAMI cell medium.
The following day, media containing transducing particles were collected, filtered with a 0.45-lm syringe filter, and added to growing MIAMI cells. MIAMI cells were incubated with transducing particles overnight, and then the media were changed to
MIAMI cells media.
Flow Cytometry Analysis
About 107 cells were harvested, washed with phosphate buffered saline (PBS), and resuspended in DMEMgfp (Evrogen,
MC101, Moscow, Russia). Just before flow analysis, cells were vortexed and filtered. Samples were analyzed with the Accuri cytometer (Ann Arbor, MI, USA) using a blue laser and narrow bandpass filters. Data were analyzed using C-flow (Accuri, Ann
Arbor, MI, USA).
RESULTS AND DISCUSSION
GFPmut3* Variants Created in the E. coli Genome
To change the color of GFPmut3*, bacteria transiently expressing Beta were electroporated with mutagenic oligos encoding the desired substitution of one or more nucleotides near the chromophore sequences of the gfpmut3* gene. Recombinants were identified by a change in colony fluorescence or by allelespecific PCR. Protein extracts prepared from isolates of each recombinant class were analyzed by fluorescence spectroscopy
(Fig. 1). We corroborated that changes in the chromophore region of GFPmut3*(F64G65Y66. . .T203) produced spectral changes from green fluorescence. GFPmut3*(F64G65W66. . .T203) produced a blue-shifted variant (Violeta). GFPmut3*(F64T65W66. . .T203) and
GFPmut3*(L64T65W66. . .T203) produced cyan variants (Aqua and
Mar, respectively). GFPmut3*(F64G65Y66. . .Y203) produced a yellow variant (Amarillo), whereas GFPmut3*(L64T65W66. . .
Y203) produced a greener cyan variant (Bronze). We determined that these variants could be distinguished by microscopy (Fig.
1a) and flow cytometry (e.g., Supporting Information Fig. 2).
Even the modestly shifted Aqua variant is easily resolved from
GFPmut3*, demonstrating the utility of oligo recombineering for producing useful variants for multicolor studies.
Recombineering Optimization
Initially, while creating the mutants using standard recombineering methods, the frequency of mutant recombinants was low

Figure 1. Fluorescent proteins created by in vitro (conventional) and in vivo (recombineering) site-directed mutagenesis. Mostaza and Azure FP were created by in vitro site-directed mutagenesis, whereas all others were created by recombineering. (A)
Confocal image of Aqua, GFPmut3*, and Amarillo E. coli. (B)
Excitation and emission spectra of FP extracts.
(\0.1%). Therefore, we evaluated how to make the protocol more efficient. In our studies, we learned that the efficiency of mutagenesis was heavily influenced by the outgrowth time following electroporation, the oligo sequence, the structure of the heteroduplex recombination intermediate, and the transformation

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Figure 2. Single-stranded deoxyribonucleic acid recombineering can be very efficient. Unless specified, strain RIK423 was recombined with the lagging-strand 99-base oligo 11. Recombination frequency is reported in each panel as the percent of recombinant colonies among the total number of colonies. (A) Segregation of recombinants during outgrowth. Sectored recombinants are colonies with a mixture of recombinant and nonrecombinant cells. Solid recombinants are colonies composed of recombinant cells only. (B)
Strand bias in targeting the lagging- or leading-strand template. Recombination targeting the lagging-strand template (oligo 11 3
RIK423) was 72 times more efficient than recombination targeting the leading-strand template (oligo10 3 RIK410). (C) The average recombineering frequency approaches 40% when normalized to transformation frequency. (D) MMR can be a potent inhibitor of recombination. MMR was evaluated in isogenic MMR1 and MMR2 (DmutS::Kan) strains. A one-tailed unpaired t-test with Welch’s correction was performed for each of the MutS1 and MutS2 pairs. Additional details are given in the Supporting Information. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] efficiency. The study of each parameter is shown in Fig. 2 and discussed below.
Following electroporation, cells are grown in liquid medium to recover before plating (14). We saw that different outgrowth times produced different apparent recombination frequencies
(Fig. 2a). Shortly after electroporation, Beta is thought to bind an oligo and anneal it to the complementary target sequence as the target is replicated (15), creating a heteroduplex recombination intermediate with the mutation present on one of the two
DNA strands. If bacteria are plated before mutant and nonmutant DNA molecules segregate, recombinant colonies are ‘‘sectored’’ with part of the colony composed of mutant cells and the remainder composed of nonmutant cells (Supporting Information Fig. 2b and ref. 14). Plating during the first hour of out-

growth allows one to accurately assess recombination frequencies and also to most efficiently identify mutants. On the other hand, if bacteria are plated after segregation, pure mutant colonies appear at a frequency equal to the initial recombination frequency divided by the number of DNA chains in the original recombinant cells (16), increasing the number of colonies one must screen to recover mutants. We found that plating cells 30 min after electroporation is an optimal balance of high viability and high recovery of recombinant (mutant) colonies.
Previous reports (e.g., ref. 17) indicate that there is a DNA strand bias in recombineering that results from the direction of replication fork travel across the target gene. To examine strand bias in our system, oligos were designed to anneal to one target strand or to its complement. Reproducibly, oligos predicted to hy-

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Figure 3. Discrimination of MIAMI human stem cells expressing eGFP and a Mostaza variant of eGFP. (A) Confocal image of
MIAMI cell variants. The bar in white at the lower right corner corresponds to 50 lM. (B) Flow cytometry of MIAMI cell variants. Excitation was from a 488-nm laser. Fluorescence emission spectra were collected using 510 6 7.5 nm and 540 6 10 nm filters. Fluorescence intensity was transformed from Cartesian to polar coordinates. Each dot represents one cell.

bridize to the lagging-strand template produced $ 70 times more mutant recombinants than oligos targeted to the leading-strand template (an example is shown in Fig. 2b). Therefore, we recommend using oligos complementary to the lagging-strand template.
The heteroduplex recombination intermediate contains one or more mispaired bases. DNA mismatch repair (MMR) can be a potent inhibitor of recombination, depending on the identity and number of mispaired bases in the heteroduplex (17). We evaluated inhibition of FP engineering by MMR using oligos that created different heteroduplex mispairs. The mutS gene was deleted to inactivate the MMR complex (18), and recombineering experiments were carried out in isogenic MutS1 and MutS2 strains (Fig. 2d and Supporting Information Table 3). The highest recombineering rates were achieved with single nucleotide mismatches when MMR was avoided. For example, the CC mispair escapes MMR and is the most efficient intermediate for mutagenesis in MutS1 strains. Interestingly, mispairs of two to nine nucleotides are equally good substrates if MMR is avoided
(the median recombination frequencies are not different by
Kruskal–Wallis ANOVA; P 5 0.4366). We recommend oligos designed to produce the least MMR-correctable heteroduplex.
Alternatively, one may inactivate the MMR system during mutagenesis and then recover the mutated gene into an MMR1 strain to reduce the chance of picking up additional mutations from the ‘‘mutator’’ phenotype of MMR2 strains.
The efficiency of introducing oligos into cells determines the amount of substrate available for recombineering. We saw that recombination frequencies fluctuated with the transformation ef-

Table 1
Rules for high efficiency in vivo site-directed mutagenesis by recombineering
Rule

Advice

One

Use dividing cells to enrich for replication intermediates. Use oligos complementary to the lagging-strand template. Use oligos that incorporate a CC mismatch in the paired recombination intermediate. Alternatively, use a 21 nucleotide mismatch or knock out
MMR.
Plate transformants within 30–60 min of outgrowth.

Two
Three

Four

ficiency of individual cell preparations. We saw a positive correlation (by a one-tailed nonparametric correlation test; P 5
0.0215) between the efficiency of plasmid uptake (to estimate transformation) and recombineering. We found that working with cells in early log phase improved both transformation and recombineering efficiencies.

eGFP Variants Created in a Lentiviral Plasmid
To extend the utility of FP engineering to mammalian cells, we modified egfp carried in a lentiviral plasmid propagated in
E. coli. Recombineering in vivo or site-directed mutagenesis in

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vitro was performed as described in Supporting Information
Methods and FP variants identified by altered bacterial colony fluorescence. We obtained eGFP(L64T65H66. . .T203) with a pronounced blue spectral shift (Azure), eGFP(L64T65W66. . .T203) with cyan spectra (Celeste), and eGFP(L64T65Y66. . .Y203) with yellow spectra (Mostaza), as shown in Fig. 1. We generated lentiviral stocks of the engineered FP variants and used them to transduce human MIAMI stem cells. As shown in Fig. 3,
MIAMI cells expressing Mostaza FP are easily distinguished from cells expressing eGFP by microscopy and flow cytometry.
The differences allow excellent quantitation over several orders of magnitude in mixtures (Supporting Information Fig. 3).

CONCLUSION
We have demonstrated that FP sequence changes easily introduced in vivo via recombineering produce significant spectral shifts. We showed that this method is useful for generating multicolor reporters and novel FPs. The Bronze variant looks especially promising for imaging studies, as the excitation maximum (468 nm) overlaps a common laser wavelength. This approach is not restricted to modifying FP genes in the E. coli genome, but also works for altering FP reporter genes in plasmid and viral sequences propagated in E. coli.
Using the optimized protocol, the efficiency of FP engineering is $ 40% of transformed cells (Fig. 2c and Supporting Information Table 3). Therefore, the optimized protocol for in vivo mutagenesis via recombineering is nearly as efficient as in vitro site-directed mutagenesis but without size limits on target
DNA molecules. A detailed protocol with explanations is provided in the Supporting Information Methods. The most critical parameters for successful mutagenesis via recombineering are summarized in Table 1.

ACKNOWLEDGEMENTS
The authors thank Dr. George McNamara of the UM Analytical
Imaging Core Facility and Dr. Pedro Salas for advice and assistance. They thank Dr. Donald L. Court, Dr. Jakob Reiser, Dr.
Chun Chau Sze, Dr. Priya Rai, and Dr. Kenneth E. Rudd for cell lines, plasmids, or bacterial strains and Dr. Guy Howard for other materials and advice. Support for this work was provided by (IMSD and F31-GM089125-01 fellowships to MVC), the
UM Interdisciplinary Stem Cell Institute (QH), from the VA
GRECC (PS) and from the Leadership Alliance, Eli Lilly, and the UM Developmental Center for AIDS Research (P30 AI
073961 to RSM).

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