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High-efficiency Scarless Genetic Modification in 2

Escherichia coli Using Lambda-Red Recombination and I-SceI 3

Cleavage 4

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Method for Bacterial Genetic Modification 6

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Junjie Yang1,2, Bingbing Sun1,2, He Huang1,2, Yu Jiang1,2, Liuyang Diao1,2, Biao Chen1,2, 8

Chongmao Xu1,2, Xin Wang1,3, Jinle Liu1,3, Weihong Jiang1, Sheng Yang1,2# 9

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1 Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai 11

Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China 12

2 Shanghai Research Center of Industrial Biotechnology, Shanghai 201201 , China 13

3 University of the Chinese Academy of Sciences, Beijing 100080, China 14

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# Correspondence to: Sheng Yang, 300 Fenglin Road, Shanghai 200032, China; Tel: 17

+86-21-54924173; Fax: +86-21-54924015; Email: syang@sibs.ac.cn; 18

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AEM Accepts, published online ahead of print on 18 April 2014Appl. Environ. Microbiol. doi:10.1128/AEM.00313-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 20

Genetic modifications of bacterial chromosomes are important for both fundamental and 21

applied research. Here, we developed an efficient, easy-to-use system for genetic modification 22

of the Escherichia coli chromosome. The system uses two plasmids, lambda (λ)-Red 23

recombination and I-SceI cleavage. An intermediate strain is generated by integration of a 24

resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus using 25

λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the 26

target gene fragment with the desired modification flanked by I-SceI recognition sites, together 27

with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI 28

cleavage of the chromosome and donor plasmid allows λ-Red recombination between 29

chromosomal breaks and linear double strand DNA from the donor plasmid. Genetic 30

modifications are introduced into the chromosome and the placement of the I-SceI sites 31

determines the nature of the recombination and the modification. This method was 32

successfully used for cadA knock-out, gdhA knock-in, pepD seamless deletion, site-directed 33

mutagenesis of the essential metK gene, and replacement of metK with Rickettsia 34

S-adenosylmethionine transporter gene. This effective method can be used with both essential 35

and nonessential genes modification, and will benefit basic and applied genetic research. 36

Key words: I-SceI endonuclease; lambda Red recombination; genetic modification; 37

markerless; Escherichia coli 38

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INTRODUCTION 41

Lambda (λ)-Red recombination, which triggers recombination between a bacterial 42

chromosome and foreign, linear double-stranded DNA (dsDNA), is a common technique in 43

Escherichia coli for genetic modification (1-4). The λ-Red recombination method relies on 44

three proteins: 5′→3′ exonuclease exo, the single-stranded DNA (ssDNA)-binding protein beta, 45

and the degradation inhibiting protein gam (4). The λ-Red recombination replaces a specific 46

chromosomal sequence with a selectable antibiotic resistance gene flanked by homologous 47

arms. Homologous recombination is efficient for linear DNA with homologous arms of 48

approximate 40 or more bp (2, 3). For markerless genetic modification, the antibiotic 49

resistance cassette integrated into the chromosome must be removed. Common methods for 50

markerless genetic modification are based on the Cre-loxP or Flp-FRT systems (3, 5). These 51

methods leave a single loxP or FRT scar sequence at the chromosomal locus, which can have 52

undesirable consequences such as native-locus gene replacement or mutagenesis. If a 53

second modification site is close to a former site, recombination can occur between the loxP or 54

FRT sequences. 55

Counterselection is effective for generating genetic modifications without leaving 56

selectable markers or scars. Published protocols are usually based on two successive rounds 57

of recombination: (1) integration of a positively selectable marker (e.g., an antibiotic resistance 58

gene), and (2) selection for marker loss (counterselection). Counterselectable markers include 59

Bacillus subtilis SacB (6), E. coli galactokinase (galK) (7) and thymidylate synthase A (thyA) (8, 60

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9), streptomycin resistance (10) and the fusaric acid sensitivity system (11). These methods 61

have several disadvantages including: (1) some counterselectable markers, such as SacB, 62

must be combined with a positively selectable marker, which increases cassette length; and (2) 63

some counterselectable markers as galK or thyA can be used only in host strains with a 64

particular auxotrophy. 65

The yeast Saccharomyces cerevisiae endonuclease I-SceI is a novel tool for unmarked 66

chromosomal manipulation. I-SceI is an intron-encoded endonuclease with an unusually long 67

recognition sequence of 18 bp (12, 13). Bacterial genomes usually do not have natural I-SceI 68

recognition sites. If an I-SceI recognition site is introduced into a bacterial genome, the 69

expression of I-SceI endonuclease induces lethal double-strand breaks (DSBs) within the 70

genome, inhibiting DNA propagation and cell growth. Several methods for site-directed 71

mutagenesis of BACs or bacterial genomes that use I-SceI expression are reported (14-17). 72

These methods are based on two successive rounds of recombination, similar to other 73

counterselection systems. The approaches can be categorized into two types (18). The first 74

type requires integration of a duplicated sequence with the I-SceI recognition site sequences 75

in a first round of recombination. The duplicated sequence is as a substrate for recombination 76

in the second round (16, 17, 19). This type of approach is efficient, but requires complex 77

design and construction. The second type of approach includes two rounds of separated 78

recombination. Blank, et al. reported a method for scarless mutagenesis of the Salmonella 79

enterica chromosome (20). Resistance marker gene and the I-SceI recognition site(s) are 80

integrated into genome, and then dsDNA fragment with modification replaces the resistance 81

marker gene. Kuhlman and Cox reported another method that integrates large fragments into 82

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the E coli genome (14, 15). Resistance gene flanked by I-SceI recognition sites and 25bp 83

exogenous sequences for “landing pads” are inserted into the chromosome. Then a fragment 84

carrying insertion is excised by I-SceI from a donor plasmid and incorporated into the genome 85

via recombination at the landing pads (14, 15). 86

Another technique, “Gene Gorging”, also uses two plasmids, λ-Red recombination and 87

I-SceI cleavage. I-SceI cleavage of the donor plasmid creates a linear dsDNA substrate, which 88

would replace the wild type chromosomal allele. "Gene Gorging" frequencies for precise 89

mutations in the E. coli genome are not sufficiently high, only 1-15%, limiting its use (21). 90

In this study, a new, two-plasmid method for unmarked genetic modification of E. coli was 91

established. The method uses λ-Red recombination and I-SceI cleavage. E. coli endogenous 92

sequences were used as substrates for recombination and no scar was introduced into 93

genome. The new method was successfully used for gene deletion and insertion, seamless 94

gene deletion, site-directed mutagenesis, and gene replacement. The new method could be 95

used to modify both nonessential and essential genes. 96

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MATERIALS AND METHODS 98

Strains, plasmids and growth conditions 99

Strains and plasmids used in this study are in Table 1. E. coli strains MG1655 and BL21 100

(DE3) were used for genetic modification experiments; strain DH5� was used for recombinant 101

DNA manipulations. All E. coli strains were cultured in LB (Luria-Bertani) medium [1% tryptone 102

(Oxoid or Angel Yeast), 0.5% yeast extract (Oxoid or Angel Yeast), 1% NaCl] supplemented 103

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with 100 mg/L ampicillin, 50 mg/L kanamycin, 100 mg/L spectinomycin, 100mg/L apramycin or 104

1 mmol/L S-adenosylmethionine (SAM), as required. 105

Oligonucleotides used are in Table S1. PCR was performed using Taq (Fermentas), 106

KOD-plus-neo or KOD-FX (Toyobo) polymerase. I-SceI endonuclease and restriction enzymes 107

were purchased from Takara or Fermentas. 108

Modular plasmids for donor and templates 109

A 1.5 kb NotI fragment containing the kanamycin -resistance gene kanMX from pUG6 (22) 110

was cloned into pBluescript II KS (-) (23) yielding pKS-K. A 1.7 kb fragment containing the 111

kanMX gene was amplified from pKS-K using primer pairs T3_IsceI/T7_IsceI and digested 112

with BssHII. The resulting fragment was used to replace a 0.2 kb BssHII region containing the 113

multiple cloning sites (MCS) of pBluescript II KS (-). The resulting ampicillin-resistance and 114

kanamycin-resistance plasmids were pKSKI-1 and pKSKI-2 and differed in insert direction. 115

pKSKI-2 was digested with NotI to excise kanMX and the plasmid backbone was recircularized 116

with T4 DNA ligase, yielding pKSI-1. 117

Apramycin-resistance or spectinomycin-resistance genes on 1.5 kb fragments were 118

amplified from pIJ773 and pIJ778 (24), and T-A cloned into pMD18-T simple (Takara) for 119

plasmids pMDIAI and pMDISI. 120

Bifunctional helper plasmids 121

The rhaB promoter fragment and the I-SceI encoding region fragment from pUC19RP12 122

(19) were joined by overlapping PCR and cloned into pKD46 (3) at the NcoI site for plasmid 123

pREDIA, which was similar to previous reported pREDI plasmids (16) with some differences in 124

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the I-SceI-coding region. 125

The ampicillin-resistance gene of helper plasmids was replaced with the 126

kanamycin-resistance gene from pPIC3.5K (Invitrogen). Bifunctional helper plasmids 127

pREDTAI and pREDTKI with the trc promoter were constructed using the process used for the 128

rhaB promoter. The trc promoter was amplified from pTRC99a (25). (See Supplementary 129

Material for detail process of plasmid construction, available online) 130

Modifications in nonessential genes 131

A linear fragment with resistance genes flanked by I-SceI recognition sites was introduced 132

by electroporation into MG1655 using conventional Red-mediated recombination (3) to obtain 133

an intermediate strain. A bifunctional helper plasmid or the Red helper plasmid pKD46 were 134

used for this step. Transformants were tested by colony PCR to confirm integration of the 135

resistance gene and I-SceI recognition sites. After transformation, the bifunctional helper 136

plasmid remained for further recombination and the conventional Red helper plasmid pKD46 137

was cured by 42°C growth (3). 138

Donor plasmid based on the pKSI-1 vector or conventional T-vectors (Takara) harboring 139

modifications were constructed by traditional restriction cloning, overlapping PCR, or Gibson 140

assembly (26). (See Supplementary Material for detail process of donor plasmid construction, 141

available online) Donor plasmids were transformed into intemediate strains; or donor 142

plasmids and bifunctional helper plasmids were co-transformed into helper-plasmid-free 143

intermediate strains. 144

Resulting colonies were incubated in LB medium with 0.5% glucose and 50 μg/mL 145

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ampicillin and kanamycin (selection for both the donor and the helper plasmid) for 6–8 hours or 146

overnight; 40 µl seed culture was used to inoculate a test tube with 4 mL LB with 10 mmol/L 147

L-arabinose and 50 μg/mL ampicillin or kanamycin (selection for the helper plasmid only). After 148

30 ºC and 200 rpm for ~2 h, L-rhamnose or IPTG (isopropyl β-D-1-thiogalactopyranoside) was 149

added to 20 mmol/L with cultivation at 30 ºC for 6–8 hours or overnight. From this culture, 40 µl 150

were used to inoculate 4 mL LB medium with 20 mmol/L L-rhamnose or IPTG, 10 mmol/L 151

L-arabinose, and 50 μg/mL ampicillin or kanamycin (selection for the helper plasmid only), 152

which was cultivated at 30 ºC for 6–8 hours or overnight. Cells were harvested, suspended in 153

sterile water, diluted (10-1 to 10-4) and spread on LB plates with 20 mmol/L L-rhamnose or IPTG, 154

10 mmol/L L-arabinose and 50 μg/mL ampicillin or kanamycin (selection for the helper plasmid 155

only). Controls were LB plates with apramycin or spectinomycin, L-rhamnose or IPTG, 156

L-arabinose, and ampicillin or kanamycin spread at the same time. (Alternatively, after the first 157

~2 h L-arabinose inducing Red enzymes, arabinose-free medium with only L-rhamnose or 158

IPTG for inducing I-SceI and antibiotic for maintaining helper plasmid could be used, to avoid 159

the potential deleterious effects caused by the prolonged expression of the lambda Red 160

functions; (27) but a slightly decreased efficiency would be obtained. ) After overnight at 30 ºC, 161

colonies were analyzed for apramycin or spectinomycin sensitivity and final clones were 162

confirmed by colony PCR. (See Supplementary Material for a step-by-step protocol, available 163

online) 164

Modifications in metK 165

Using Red-mediated recombination(3), an apramycin resistance gene and I-SceI 166

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recognition sites (IsceI-aprR-IsceI cassette) was inserted into the yqgC site, and a 167

spectinomycin resistance gene and I-SceI recognition sites (IsceI-spcR-IsceI cassette) was 168

inserted into galP site, by successive electroporation steps for strain A10. yqgC and galP are 169

nonessential genes next to the essential metK gene. 170

A 2.5 kb PCR fragment containing part of the speA gene, full-length yqgB-yqgC-metK 171

genes and part of the galP gene was cloned into pKSI-1 for pKSI-metK. Site-directed 172

mutagenesis of metK followed the protocol in QuikChange Site-Directed Mutagenesis kits 173

(Agilent) to obtain the donor plasmid pKSI-metKmut (28). A 1 kb fragment with the gene for the 174

SAM transporter (29, 30) was synthesized and replaced the metK coding region in pKSI-metK, 175

yielding pKSImetKSamT. 176

Mutagenesis or replacement of metK was the same as for the nonessential genes, with 1 177

mmol/L SAM added to LB medium if required. For replacing metK gene with the Rickettsia 178

SAM transporter gene, clones were also analyzed for SAM auxotrophy. 179

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RESULTS 182

Scarless modification method strategy 183

This method included a conventional initial step and a final recombination step (Figure 184

1AB). In the initial step, a resistance gene flanked by I-SceI recognition sites was inserted into 185

the chromosome by conventional λ-Red PCR targeting (Figure 1A); meanwhile, a fragment 186

carrying the desired modification was inserted into donor plasmid with I-SceI recognition sites. 187

In the final recombination step, expression of I-SceI endonuclease induces DSBs within both 188

the genome and the plasmid. The fragment with modification is excised from a donor plasmid, 189

and then incorporated into the genome at the DSB locus via λ-Red recombination. (Figure 1B) 190

Three groups of plasmids (bifunctional helper plasmid, templates plasmid and modular 191

donor plasmid) were constructed (Table 1, Figure 2) for performing this method. The plasmids 192

have been distributed to Addgene (http://www.addgene.org/) with IDs 51625~51628, 193

51652~51655 and 51725 . 194

The method could be initiated by transformation of a host strain with a bifunctional helper 195

plasmid (Figure 1A)., which were constructed from the λ-Red helper plasmid pKD46 (3). The 196

λ-Red genes gam, bet and exo were driven by the araBAD promoter and inducible with 197

L-arabinose (Figure 2A). These genes mediated recombination between target regions and 198

homologous arms. Plasmids with the I-SceI gene under the control of the trc or rhaB promoter 199

were inducible with IPTG or L-rhamnose. The temperature sensitivity of the pSC101 200

replication origin maintained plasmids at low copy number and for easy curing by growth at 201

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42°C. Two versions of the helper plasmids were differentiated by use of either the kanamycin 202

or ampicillin resistance markers. This design made the plasmids compatible with different 203

donor plasmids (Figure 2A). 204

Plasmids pMDIAI and pMDISI were used as PCR templates to amplify 205

apramycin-resistance or spectinomycin-resistance genes flanked by I-SceI recognition sites. 206

(Figure 2C). The small construct sizes allowed simple and reliable integration of resistance 207

marker genes into any location using established λ-Red PCR-targeting recombination 208

methods.(3) (Figure 1A). 209

To facilitate donor plasmid construction, several modular vectors were constructed based 210

on the high-copy vector pBluescript II KS (-) (23). Plasmid pKSI-1 consisted of a 211

pBluescript II KS (-) backbone, an MCS, and two I-SceI recognition sites (Figure 2B). The 212

MCS could be used for further subcloning steps. Plasmids pKSKI-1 and pKSKI-2 were similar, 213

with a kanamycin- resistance gene kanMX in the MCS. Alternatively, donor plasmids could be 214

constructed with common high-copy T-vectors such as pMD19-T simple and pBackZero-T 215

(Takara). In this case, I-SceI recognition sites were introduced by a PCR step using longer 216

primers. Donor plasmids based on pKSI-1 or common T-vectors showed high recombination 217

frequencies (Table 2). 218

Markerless knockout and knock-in of selected genes 219

knock-out of cadA 220

An apramycin-resistance gene cassette flanked by I-SceI recognition sites was integrated 221

into the cadA locus by λ-Red PCR targeting to obtain an intermediate strain. Positive clones 222

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were screened by PCR (Figure 3A). PCR products were digested with I-SceI, to check for 223

functionality of the inserted I-SceI restriction sites (data not shown). 224

To construct a donor plasmid, truncated cadA segments with I-SceI recognition sites was 225

cloned into a pBackZero-T vector. The donor plasmid was transformed into the intermediate 226

strain for the final recombination step. In the recombination step, λ-Red enzymes, then I-SceI 227

were induced, leading to cleavage of both the donor plasmid and the chromosome. Once 228

DSBs were generated by I-SceI, recombination between the resulting donor fragment and the 229

DSB chromosomal locus was mediated by λ-Red enzymes (Figure 1B). 230

After growth in IPTG or L-rhamnose to induce I-SceI expression with L-arabinose to induce 231

expression of λ-Red enzymes, most surviving cells that were apramycin sensitive were 232

confirmed as having the desired deletion. The deletion was confirmed by PCR, restriction 233

endonuclease analysis and sequencing (Figure S2A). 234

Meanwhile, in the negative control experiments without donor plasmid, the frequency of 235

apramycin sensitivity of the surviving cells was lower than 5% of the total, indicating the 236

knock-out of cadA would occur by repair with the donor DNA, not by non-homologous 237

end-joining of the double stranded breaks. 238

Knock-in of gdhA 239

Results for knock-in of the gdhA gene were similar to results for knocking out cadA. In the 240

recombination step, I-SceI were induced, leading to cleavage in the chromosome at gntT gene 241

locus and evict spectinomycin-resistance gene. Once DSBs were generated at gntT locus, 242

recombination happened between the donor fragment with gdhA and the DSB chromosomal 243

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locus, mediated by λ-Red enzymes. After recombination, the gdhA gene fragment replaced the 244

spectinomycin-resistance gene. The resulting strain had the desired modification of gdhA 245

integrated into the chromosome (Figure S2B). 246

Multiple modifications 247

We also inserted the gdhA gene into the chromosome of a cadA-disrupted E. coli strain. 248

Similar modification efficiency was obtained (data not shown). The bifunctional helper plasmid 249

for cadA disruption was retained, and directly used for the PCR targeting experiments to 250

integrate spectinomycin-resistance gene into gntT locus. These results demonstrate the 251

protocol could be used repeatedly, to make multiple modifications in a strain. 252

253

Seamless deletion of pepD 254

To determine if the new method could be used for seamless deletion of a selected gene, 255

we deleted the pepD gene following a procedure similar to the process described above. An 256

apramycin-resistance gene cassette flanked by I-SceI recognition sites was integrated into the 257

pepD locus, replacing 600 bp in the pepD ORF (Figure 3A). 258

To construct a donor plasmid, upstream and downstream fragments for the pepD gene 259

were joined and subcloned into pKSI-I. The fusion fragment was suitable for seamless deletion 260

of pepD (Figure 3B). The apramycin-resistance gene fragment and remaining 1.0 kb pepD 261

fragments were replaced by “nothing” (Figure 3B). The 1.6 kb pepD ORF was seamlessly 262

deleted from the ATG start codon to the TAA terminator codon (Figure 3C). 263

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Modifications in the essential metK gene 264

To use the new method to modify an essential gene locus, a novel strategy for an 265

essential gene was designed. Two pairs of I-SceI sites with 2 different resistance genes were 266

inserted into the loci next to the target gene by consecutive λ-Red PCR targeting. Meanwhile, 267

donor plasmid with fragment carrying the desired modification was constructed as same 268

process for the non essential genes. In the final recombination step, the wild-type target gene 269

and the two resistance genes were removed from the chromosome. Then the fragment with 270

modification is excised by I-SceI from a donor plasmid and incorporated into the genome at the 271

gene locus via recombination. (Figure 4) 272

To determine if the new method could be used to modify an essential gene locus, we used 273

the method to modify the metK gene. E. coli metK is essential because it produces the only 274

enzyme for synthesis of SAM, which is involved in many metabolic reactions, and E. coli 275

cannot take up SAM from medium(31). As an essential gene, metK could not be directly 276

deleted, but mutations can be made in its coding region. The metK gene can be replaced by a 277

Rickettsia SAM transporter gene and the resulting cells take up extracellular SAM and survive 278

with SAM in the medium (29, 30). 279

Site-directed mutations in metK 280

The intermediate strain A10 was constructed with two resistance genes with 2 pairs of 281

I-SceI sites inserted into the loci next to metK (Figure 4A). A donor plasmid was constructed 282

with mutagenesis fragments containing metK sequences with three synonymous mutations. 283

In the recombination step, I-SceI cut at the 2 pairs of I-SceI sites next to metK, and the 284

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wild-type metK gene and the two resistance genes were removed from the chromosome. 285

Subsequently, the mutation fragment from the donor plasmid replaced the wild-type fragment 286

via homologous recombination, introducing the mutations into the metK ORF without scar 287

fragments (Figure 4B). Mutations were confirmed by PCR, restriction endonuclease analysis 288

and sequencing (Figure 4C). 289

Replacement of metK with Rickettsia SAM transporter 290

A donor plasmid containing the SAM transporter coding region, the metK promoter and 291

the metK terminator was constructed. The plasmid was transformed into the A10 intermediate 292

strain. Using the same procedure, the SAM transporter fragment was used to replace wild-type 293

metK via homologous recombination (Figure 4 B,C). The resulting strains had SAM 294

auxotrophy and grew in medium supplemented with SAM (Figure S1). 295

Curing plasmids 296

Donor plasmids are efficienty cured by I-SceI cleavage (14). After recombination, at least 297

half of colonies with the desired genotype were sensitive to ampicillin or kanamycin (Table 2), 298

showing efficient curing of donor plasmids. For the colonies with the desired genotype and the 299

donor plasmid remaining, prolonged culture in IPTG or L-rhamnose medium to induce I-SceI 300

expression resulted in donor plasmid curing (data not shown). Helper plasmids can be cured 301

by 42°C growth (3). 302

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Method efficiency 304

Preparatory steps followed an established λ-Red PCR targeting method(3). Integration 305

frequencies (colonies with expected PCR bands per tested spectinomycin/apramycin resistant 306

colonies) were greater than 50%, depending on the size of the homologous regions and the 307

targeted genomic locus (data not shown). 308

For recombination for modification and dominant marker recycling, IPTG induction of the 309

trc promoter to stimulate I-SceI expression gave higher recombination frequencies and donor 310

plasmid curing frequencies than rhaB promoter. (Table 2). 311

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315

DISCUSSION 316

The scarless genetic modification strategy described here was based on a simple 317

two-plasmid procedure using λ-Red recombination and endonuclease I-SceI cleavage. A 318

fragment with I-SceI recognition sites was inserted into the target site using λ-Red 319

recombination and positively selected by conventional λ-Red PCR targeting (2, 3). I-SceI 320

cleaved both the donor plasmid and the chromosome. Incorporation of the insertion fragment 321

was enhanced by the expression of λ-Red enzymes and led to integration of the donor 322

fragment and scarless genetic modification (Figure 1). Generally, the strategy took about 1.5-2 323

weeks, with donor plasmid construction and λ-Red PCR targeting procedure performed 324

simultaneously. We used this strategy in both E. coli K-12 strain MG1655 and the B strain 325

BL21(DE3). This strategy should be applicable in any bacterial strains that can be modified 326

with λ-Red or other PCR targeting recombination systems. As endonuclease I-SceI directly 327

cleavage at the chromosome, this method would be applicable for host strains with any 328

genotypes, including those with genotypes not suitable for published counterselectable 329

markers. 330

Our method use two separated rounds of recombination, and this approach is also used 331

by some similar previously reported methods (14, 15, 20). This approach avoids the 332

integration of a duplicated sequence in a first recombination to be a substrate for the second 333

recombination (16, 17) and gives flexibility in designing and performing experiments. As shown 334

by our pepD seamless deletion, in the first PCR-targeting step, short arms homologous to 335

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pepD ORF were used for the first recombination, deleting a pepD coding region of 336

approximately 600 bp by replacement with a resistance marker gene and leaving pepD coding 337

region of approximately 1 kb. Subsequently, fragments homologous to upstream and 338

downstream regions of pepD were used for the second recombination, resulting in complete 339

deletion of the truncated pepD segments and the resistance genes from the start codon to the 340

terminator codon (Figure 3). 341

Our method shows some similarity to a method described previously by Kuhlman and Cox 342

for integrating large fragments into the E coli genome (14, 15). Kuhlman and Cox‘s method 343

requires integration of 25bp exogenous sequences into the genome to generate a “landing 344

pad”, so it is not a seamless method. Our method uses the endogenous fragment for 345

recombination in both initial and the final recombination steps. No redundant sequences are 346

introduced into the genome, so seamless modification is possible. 347

Our method could be used for both nonessential and essential genes. Of the total 4288 E. 348

coli genes, 3985 are nonessential and 303 genes are essential candidates including 37 with 349

unknown function (1). To modify nonessential genes, I-SceI recognition sites and resistance 350

gene could be inserted directly into the gene (Figure 1). For modifying essential genes, the 351

I-SceI recognition site and resistance genes could be inserted upstream and downstream of 352

the essential gene (Figure 4). In both cases, subsequent recombination was accurate as 353

expected. 354

As previously reported, in vivo cleavage of donor plasmids by I-SceI expression has 355

advantages over introduction of linear DNA fragments by direct transformation(14). Repair and 356

editing mechanisms during chromosomal and plasmid replication result in high fidelity. The 357

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higher transformation efficiencies of supercoiled plasmids facilitates the experiments. High 358

plasmid copy numbers are important for maintaining a high concentration of intracellular 359

fragments (14). The gene gorging method is reported to result in chromosomal modification 360

frequencies as high as 1–15%, even without cutting chromosomes (21). 361

I-SceI expression must be considered. In our experiments, using the rhaB promoter for 362

I-SceI expression resulted in about a half of the colonies with the resistant phenotype. PCR 363

confirmed that no mutations occurred within or next to the resistance genes and the integration 364

site (data not shown). The trc promoter gave a higher efficiency for fragment replacement and 365

donor plasmid curing, probably because of differences in expression levels from the different 366

promoters (32). 367

Our method could be a simple and easy way to move mutations from one strain into 368

another. For example, generating a strain with a desired phenotype generated by random 369

mutagenesis or evolution require introducing every mutation into a wild-type strain to analyze 370

the effect on function (33). Such mutations can be identified by high-throughput genome 371

sequencing and confirmed by PCR and dideoxy chain-termination sequencing. The pKSKI 372

series could be made into T-vectors by simple digestion (34-36), so donor plasmids could be 373

constructed by direct T-A cloning. A resistance gene cassette and I-SceI recognition sites 374

could be integrated into the gene locus of the recipient strain by λ-Red PCR targeting. The 375

fragment from the donor plasmid harboring the mutation could be integrated into the genome 376

of recipient strains to replace the wild type allele. 377

Another advantage of our new method is that different modifications could be performed 378

on a single intermediate strain. By using different donor plasmid, a target gene of interest could 379

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be modified differently. Any methods could be used to construct the donor plasmid, even by 380

artificial whole gene synthesis. The only requirement is inclusion of upstream and downstream 381

homologous regions and I-SceI recognition sites. According to our experiments, homologous 382

regions in 300~500bp length could benefit both plasmid construction and recombination 383

efficiency. The designed sequence on the donor plasmid will be the exact sequence introduced 384

in the final strains. 385

The system could potentially be improved. For making multiple modifications, a different 386

set of resistance marker genes flanked by I-SceI recognition sites could be integrated 387

successively into target sites. Donor fragments for each modification could be assembled 388

together with I-SceI sites between the fragments. The assembled fragments could be cloned 389

into a single donor plasmid. Upon I-SceI cleavage of the chromosome and donor plasmid, the 390

linear dsDNA fragments from the donor plasmid could introduce a series of genetic 391

modifications into the chromosome. Recent studies demonstrated that Cas/CRISPR 392

RNA-guided targeting methods can be used to edit bacterial genomes (37). These methods 393

appear to be more efficient than previously reported TALENs method (38). Two-plasmid 394

recombination methods could be further modified by replacing I-SceI cleavage with 395

Cas/CRISPR and combining two-step recombination into one step. 396

Our data demonstrated that the new method presented here are a useful tool for 397

unmarked genetic modification of E. coli. This effective method can be used with both 398

essential and nonessential genes modification, and will benefit basic and applied genetic 399

research. 400

401

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402

ACKNOWLEDGEMENTS 403

We thank Dr. Daibang Nie and Dr. Jian Li for thoughtful discussions and critical reading of 404

the manuscript. We thank Prof. Benjamin S Glick (University of Chicago) providing SnapGene 405

software for producing plasmid maps. We also thank the editor and the anonymous referees 406

for their comments and constructive suggestions. 407

This work was supported by The National Basic Research Program (973 Program) of 408

China (2014CB745100, 2011CBA00800). 409

Conflict of interest statement: None declared. 410

411

412

SUPPLEMENTAL MATERIAL 413

Supplementary Table S1, Supplementary Figures S1~S2, Supplementary Methods, 414

Protocol. 415

416

REFERENCES 417

418

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522

523

524

525

526

527

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528

Figure Legends 529

530

Figure 1. Diagram of this two-plasmid method. 531

A: Antibiotic cassette fragment with I-SceI recognition sites integrated at a target via λ Red 532

-mediated recombination creating an intermediate strain. B: Intermediate strain with mutation(s) 533

and I-SceI recognition sites was transformed with donor plasmid. Expression of I-SceI was 534

induced by L-rhamnose or IPTG (isopropyl β-D-1-thiogalactopyranoside). I-SceI recognition 535

sites in donor plasmid and chromosome were cleaved. Integration of donor fragment at the 536

cleaved site of chromosome was mediated by λ Red recombination. 537

538

Figure 2. Plasmids used for this method. 539

A: pREDTKI and pREDTAI (helper plasmids) with arabinose-inducible (araB promoter ) 540

λ-Red recombinase functions, IPTG-inducible (trc promoter ) I-SceI expression. B: pKSI-1 541

(modular plasmids for donor) based on the high-copy vector pBluescript II KS (-), with multiple 542

cloning site (MCS) and two I-SceI recognition sites. C: Part of plasmid pMDIAI (template 543

plasmid) with an apramycin-resistance gene flanked by FRT (Flippase Recognition Targe) 544

sites and I-SceI recognition sites. For pMDISI, apramycin-resistance was replaced with 545

spectinomycin-resistance genes. Pink arrow: binding sites for primers MDF and MDR. 546

547

Figure 3. Seamless pepD deletion and PCR analysis 548

A: 40 bp short arms homologous to pepD ORF; 600 bp pepD segment replaced by 549

marker. B: Arms with upstream (U) and downstream (D) homology to pepD, with truncated 1.0 550

kb pepD segments and apramycin (apr) resitance genes seamlessly deleted from ATG to TAA. 551

C: PCR of pepD. BL21(DE3) (wild-type pepD), 2.5 kb; intermediate strain (pepD::apr), 3.3 kb; 552

final strain (ΔpepD), 0.9 kb. 553

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554

Figure 4. Modifications to metK gene and PCR analysis. 555

A: IsceI-apr-IsceI cassette inserted into yqgC and IsceI-spc-IsceI cassette inserted into 556

galP. YqgC and galP are nonessential genes next to metK. B: Wild-type metK and resistant 557

genes replaced by mutated metK or SAM transporter. galP fragment and speA-yqgB-yqgC 558

fragment were homologous arms. C: PCR analysis of metK. MG1655 (wild-type metK), 2.8 kb; 559

intermediate strain A10 (yqgC::apr, galP::spc), 5.9 kb; final strain with mutated metK, 2.8 kb, 560

the same length as wild-type metK; final strain with metK replaced by SAM transporter, 2.6 kb. 561

562

563

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1

orfA

marker

marker

marker

mut

mut

E. coli Chromosome

orfA 3'orfA 5' I-SceII-SceI

helper

helper

helper

helperorfA 5' orfA 3'

Double Crossover Recomination

Curing

Double Crossover Recomination

PCR-Targeting

B

A

mut

orfA 3'orfA 5'

I-SceI I-SceI

Donor

I-SceI Cutting + Recombination

Transformation

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Mark

er

Metk(

WT)

yqgC::a

pr

&galP::s

pc

SAM tr

ansporto

r

metK

(mut)

Mark

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5.9 k

2.8 k

2.6 k3.0 k

6.0 k

1.0 k

2.5 k2.8 k

C

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Marker pepD pepD::apr pepD

3.2 k

2.5 k

0.9 k

3.0 k

0.5 k

1.0 k

A

B

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Table 1: Strains and plasmids used in this study

Strain or plasmid Characteristics Source

Strains

DH5alpha General cloning host strain Takara

MG1655 The "wild type" E. coli K-12 strain CGSC

W3110 Another E. coli K-12 strain CGSC

BL21(DE3) An E. coli B strain with DE3, suitable for

protein expression. Invitrogen

Helper plasmids Characteristics

pREDIA ori SC101(ts), AmpR, araBAD promoter for

λ-Red, rha promoter for I-SceI

This work

pREDKI ori SC101(ts), KanR, araBAD promoter for

λ-Red, rha promoter for I-SceI

This work

pREDTAI ori SC101(ts), AmpR, araBAD promoter for

λ-Red, trc promoter for I-SceI

This work

pREDTKI ori SC101(ts), KanR, araBAD promoter for

λ-Red, trc promoter for I-SceI

This work

Template plasmids

pMDIAI AprR, AmpR, Apramycin resistance gene flanked

by FRT and I-SceI sites. pMD18-T simple

backbone

This work

pMDISI SpcR, AmpR, Spectinomycin resistance gene

flanked by FRT and I-SceI sites. pMD18-T

simple backbone

This work

Modular plasmids for donor

pKSKI-1 AmpR, KanR, pBluescript II KS(-) backbone with

I-SceI site - kanMX - I-SceI site, differed in insert

direction with pKSKI-2

This work

pKSKI-2 AmpR, KanR, pBluescript II KS(-) backbone with

I-SceI site - kanMX - I-SceI site, differed in insert

direction with pKSKI-1

This work

pKSI-1 AmpR, pBluescript II KS(-) backbone with I-SceI

site - multiple cloning sites - I-SceI site

This work

Other plasmid

pMD18-T simple AmpR, cloning vector Takara

pMD19-T simple AmpR, cloning vector Takara

pBackzero-T KmR, cloning vector Takara

pUG6 KmR vector EUROSCARF,

Guldener, et al., 1996

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pPIC3.5K AmpR, P. pastoris expression plasmid Invitrogen

pBluecriptII KS(-) Cloning vector Alting-Mees and Short,

1989

pKS-K AmpR, KanR, pBluescript II KS(-) backbone

with kanMX This work

pKD46 λ-Red helper plasmid Datsenko and Wanner,

2000,

pKD46K λ-Red helper plasmid with kanamycin

resistance This work

pMDcadA. cadA fragment subcloned into pMD18-T

simple This work

pMDcadA-ISceI-Apr Plasmid for integrating spectinomycin

resitance gene into cadA locus This work

pMDcadAL pMD18-T simple with truncated cadA

fragment This work

pBackZero-ISceI-cadA Donor plasmid for cadA deletion, pBackzero-T

backbone This work

pKSIpepD0.7k Donor plasmid for pepD deletion, derived from

pKSI-1 This work

pMD19-gntT gntT fragment subcloned into pMD19-T

simple This work

pMD19-gntT-spc Plasmid for integrating spectinomycin

resitance gene into the gntT locus This work

pMD19-gntT-gdhA Donor plasmid for integrating gdhA gene This work

pKSImetK metK fragment cloned into pKSI-1 This work

pKSImetKmut Donor plasmid for site-directed mutagenesis

of the metK This work

pKSImetKSamT Donor plasmid for replacing metK with SAM

transporter This work

AmpR: Ampicillin resistance; Km

R: Kanamycin resistance, AprR: Apramycin resistance; SpcR:

Spectinomycin resistance

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Table 2: Rates for marker gene eviction, donor plasmid curing and correct modification

Modification Helper plasmid/

promoter for

I-SceI expression

Donor plasmids

backbone

Marker eviction rate

(Apr and/or Spc

sensitive colonies per

tested colonies)

Donor plasmid curing rate

(Amp or Kan sensitive

colonies per tested

colonies)

Correct modification rate,

confirmed by PCR

(colonies with expected PCR

bands per tested colonies)

Correct modification

rate, confirmed by

PCR

fragment sequencing

cadA deletion pREDIA / rhaB pBackZero-T 4/10 3/4 3/3 3/3

cadA deletion pREDTKI / trc pMD19-T 20/20 18/20 18/20 4/4

gntT integration pREDTKI / trc pMD19-T 8/10 7/8 7/7 4/4

PepD seamless

deletion pREDKI / rhaB

pKSI-1 3/8 2/3 2/2 2/2

PepD seamless

deletion pREDTKI / trc

pKSI-1 6/8 6/6 6/6 6/6

MetK mutation pREDKI / rhaB pKSI-1 9/30 5/9 9/9 4/4

MetK mutation pREDTKI / trc pKSI-1 18/18 18/18 16/18 4/4

MetK

replacement pREDTKI / trc

pKSI-1 2/4 2/2 2/2 2/2

Amp: Ampicillin; Apr: Apramycin ; Spc: Spectinomycin.

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