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Journal of Biotechnology
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A R T I C L E I N F O A B S T R A C T
Keywords: To investigate gene function in Bacillus anthracis, a high-efficiency cloning system is required with an increased
Bacillus anthracis rate of allelic exchange. Golden Gate cloning is a molecular cloning strategy allowing researchers to simulta-
Golden Gate neously and directionally assemble multiple DNA fragments to construct target plasmids using type IIs restriction
Allelic exchange enzymes and T4 DNA ligase in the same reaction system. Here, a B. anthracis S-layer protein EA1 allelic exchange
I-SceI
vector was successfully constructed using the Golden Gate method. No new restriction sites were introduced into
this knockout vector, and seamless assembly of the DNA fragments was achieved. To elevate the efficiency of
homologous recombination between the allelic exchange vector and chromosomal DNA, we introduced an I-SceI
site into the allelic exchange vector. The eag gene was successfully knocked out in B. anthracis using this vector.
Simultaneously, the allelic exchange vector construction method was developed into a system for generating B.
anthracis allelic exchange vectors. To verify the effectiveness of this system, some other allelic exchange vectors
were constructed and gene replacements were performed in B. anthracis. It is speculated that this gene knockout
vector construction system and high-efficiency targeted gene replacement using I-SceI endonuclease can be
applied to other Bacillus spp.
1. Introduction 1981), and the Univector cloning system by Stephen Elledge’s labora-
tory (Liu et al., 1998; Liu et al., 2000). With these cloning techniques,
DNA recombination technology emerged in the early 1970s. target fragments can be simply, efficiently, and accurately fused to
Restriction digestion and ligation is the most commonly used cloning specific vectors. However, specific recombination site sequences are
method in the construction of recombinant DNA molecules. This retained and cannot be eliminated from the constructs developed using
method is one of the most basic molecular biology techniques, in which this cloning method, so extra amino acids are added into the expression
restriction endonucleases and DNA ligase are used to clone the gene(s) products if these sites are located in an expression sequence.
of interest into a vector. However, this method has its limitations. In 2008, Engler et al. reported a cloning strategy mediated by the
Specifically, when multi-target DNA fragments are ligated, they then type IIs restriction enzyme, known as the IIs cloning method or the
need to be inserted into the vector step-by-step, which can be a tedious Golden Gate cloning method (Engler et al., 2008, 2014), which has
and time-consuming procedure; in addition, restriction endonuclease been applied in many fields. In 2011, Weber and colleagues used the
sites are retained between target DNA fragments in the final construct Golden Gate cloning method to create three dTALEs to activate a re-
(Goff and Berg, 1976). porter construct (Weber et al., 2011). In 2014, Terfrüchte and cow-
Approximately 20 years ago, the development of site-specific re- orkers established a versatile Golden Gate cloning system for genetic
combination cloning technology provided a new, rapid, and effective engineering in fungi (Terfrüchte et al., 2014), and Engler and cow-
method of constructing DNA recombinants in vitro, such as the Gateway orkers used the Golden Gate cloning method to construct multigene
cloning system developed by Invitrogen (Hartley et al., 2000; Walhout constructs for plant transformation (Engler et al., 2009). In 2016, Vad-
et al., 2000), the Creator cloning system by Clontech (Sternberg et al., Nielsen and colleagues used the Golden Gate cloning method to
⁎
Corresponding authors.
E-mail addresses: con_an@126.com (T. Wang), wangdongshu@foxmail.com (D. Wang), flygogo.cool@163.com (Y. Lyu), fengel@sohu.com (E. Feng), jewly54@126.com (L. Zhu),
liucj@nic.bmi.ac.cn (C. Liu), springwyc@gmail.com (Y. Wang), liuxk007@163.com (X. Liu), wanghl@nic.bmi.ac.cn (H. Wang).
https://doi.org/10.1016/j.jbiotec.2018.02.006
Received 24 May 2017; Received in revised form 8 February 2018; Accepted 9 February 2018
Available online 10 February 2018
0168-1656/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T. Wang et al. Journal of Biotechnology 271 (2018) 8–16
2. Materials and methods 2.4. Subcloning the entry DNA fragments into the acceptor vector pKMBKI
to construct the recombinant allelic exchange vector
2.1. Materials
The scheme for subcloning the entry DNA fragments into the ac-
2.1.1. Bacterial strains and plasmids ceptor vector pKMBKI is illustrated in Fig. 2. First, the concentration of
Plasmid pKSV7 contains temperature-sensitive replication functions each plasmid (pGEM-U, pGEM-S, pGEM-D and pKMBKI) was measured
derived from pE194ts (permissive temperature, 30 °C; restrictive tem- using a NanoDrop spectrophotometer. The reaction mixture comprised:
perature, 37 °C and above; Table 1). It also contains an ampicillin re- 50 ng of each entry vector and the acceptor vector together with 2.5
sistance gene for selection in Escherichia coli, and a chloramphenicol units of BsaI enzyme, 2.25 units of T4 DNA ligase, 1 μl of CutSmart
resistance gene for selection in B. anthracis. The pMAD vector contains a buffer, 1 μl of ATP (10 mM), and ddH2O up to a final volume of 10 μl.
bgaB gene encoding a thermostable β-galactosidase from Bacillus The restriction–ligation procedure was performed in accordance with
stearothermophilus, which is expressed from a constitutive promoter the following program: The mixture was incubated in a water bath at
recognized in Gram-positive and Gram-negative bacteria. The pSET4s 37 °C for 30 min, followed by heating at 50 °C for 5 min to redigest and
vector contains a spectinomycin resistance gene. This vector could be eliminate any plasmid that might still contain a BsaI restriction site and
propagated at 37 °C in E. coli. The pGEM-T easy vector is a linearized then 80 °C for 5 min to inactivate both restriction enzyme and ligase. A
vector with a single 3ʹ-terminal thymidine at both ends. B. anthracis 5-μl restriction–ligation product was then added to 50 μl of DH5α
vaccine strain A16R (pXO1+, pXO2−; Table 1) was derived from chemically competent cells, which were subsequently incubated in an
strain A16 by UV irradiation. ice-bath for 30 min, followed by a 90-s thermal shock at 42 °C, then
another 2 min in the ice-bath, followed by 1-h incubation at 30 °C with
2.1.2. Enzymes and biochemical reagents shaking at a speed of 225 r/min. Then, 300 μl of bacterial culture was
2xEs Taq Master Mix (Dye) (Beijing ComWin Biotech, Beijing, plated on Luria–Bertani (LB) agar plates containing X-Gal (40 μg/ml)
China), Cobuddy High-Fidelity Rapid DNA polymerase (Beijing and kanamycin (50 μg/ml) and the plates were incubated at 30 °C
ComWin Biotech), Fast Digest EcoRI (Thermo Fisher Scientific, overnight. Twenty white colonies were picked, streaked onto LB agar
Rockford, IL, USA), Fast Digest BamHI (Thermo Fisher Scientific), T4 plates, and incubated at 30 °C for 10 h. Single clones were analyzed by
DNA ligase (Thermo Fisher Scientific), BsaI (New England Biolabs, colony PCR to determine whether the three entry DNA fragments had
USA), and ATP (10 mM; New England Biolabs, Beverly, MA, USA) were been inserted into the acceptor vector pKMBKI using three pairs of
used in this study. primers: eagup_F/R, eagdn_F/R and spcin_F/R. The recombinant
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T. Wang et al. Journal of Biotechnology 271 (2018) 8–16
Table 1
Bacterial strains and plasmids used in this study.
pKSV7 Shuttle vector, temperature-sensitive, Ampr in E. coli and Cmr in B. anthracis This lab(Smith and Youngman,
1992)
pMAD Shuttle vector, Apr in E. coli; Emr in B. subtilis; containing pclpB-bgaB fragment This lab(Arnaud et al., 2004)
pKMU7 Derive from pKSV7 by destroying a BsaI site in it. This work
pKD4 Containing kanamycin resistant gene This lab
pKMBK Constructed by inserting pclpB-bgaB and Kanr fragments into pKMU7 This work
pKMBKI Constructed by inserting I-SceI fragment into pKMBK This work
pKMUSD The production of “Golden Gate” cloning with the upstream part of the target gene, the spectinomycin resistance This work
cassette, and the downstream part of the target gene inserting into the vector pKMBKI
pGEM-T easy Ampr in E. coli, with single 3’-T overhangs This lab
pGEM-U Constructed by inserting eagup fragment into pGEM-T easy This work
pGEM-D Constructed by inserting eagdn fragment into pGEM-T easy This work
pGEM-S Constructed by inserting Spcr cassette fragment into pGEM-T easy This work
pSET4s Shuttle vector,Spcr both in E. coli and B. anthracis This lab (Takamatsu et al.,
2001)
pSS4332 expressing endonuclease I-SceI, Kanr in B. anthracis and Ampr in E. coli, is relatively unstable in B. anthracis This lab (Cybulski et al., 2009)
allowing rapid loss by segregation after removal of selection.
E. coli DH5α F-,ϕ80d/lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-,mk+),phoA,supE44λ-, thi-1, This lab
gyrA96,relA1
E. coli JM110 rpsL (Str R) thr leu thi-1 lacY galK galT ara tonA tsx dam dcm supE44 Δ(lac-proAB) /F′[traD36 proABlacIq This lab
lacZΔM15]
B. anthracisA16R Vaccine strain; pXO1+, pXO2- This lab
B.anthracisA16R△eag::spc A16R with eag gene knocked out and Spcr gene instead This work
Note: Ampr, ampicillin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Spcr, spectinomycin resistance; Kanr, kanamycin resistance.
Table 2
Primers used in this study.
Name Sequences
bgaB_F TGACATGATTACGAATTCTGACAgagaccGTCTAGTTAATGTGTAACG
bgaB_R AGTGCTTGCGGCAGCGTGACCAAAgagaccGCATATTATGTTGCCAAC
kan_F TCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGC
kan_R GTCGACTCTAGAGGATCCTGGGCGAAGAACTCCAGCAT
I-SceI_F CAAGCTTGCATGCCTGCAGGTCGACTAGGGATAACAGGGTAATGTCGACTCTAGAG
I-SceI_R TTCGCCCAGGATCCTCTAGAGTCGACATTACCCTGTTATCCCTAG
eagup_F TTTggtctcATGACCTTATCTGTTGATGGTGTACC
eagup_R TTTggtctcACTCCCTCCTTCAGGAATATGCTACT
eagdn_F TTTggtctcAATCGCGAATCACTATACACGAACA
eagdn_R TTTggtctcACCAACCAATCCATGCCTGCTAT
spc_F TTTggtctcAGGAGCTAGTGTTCGTGAATACATG
spc_R TTTggtctcACGATTATGCCGATAACTAG
eagin_F CAATGACAGCAGCAATGG
eagin_R TCAACATCAGCGTTACCTT
eagus_F GAAGTAGACGCAACTGATG
eagus_R GCTCTTGTAACCATTCTCC
eagsd_F GGCTGAATCTTCTCCATTA
eagsd_R CATACTAGAACTTGCACCAA
spcin_F TGATGTGAGAAGAGCCATTA
spcin_R CTCCAAGATAACTACGAACTG
pKSV7_F ATCGTTGTCAGAAGTAAGTTGG
pKSV7_R TGGCGTAATAGCGAAGAGG
Note: Underlined sequences indicate restriction sites; lower-case italic sequences indicate BsaI restriction sites.
plasmid was confirmed by DNA sequencing using an ABI Prism Model 2.6. Deletion of theeag gene from B. anthracis
3730XL DNA analyzer (Applied Biosystems, Carlsbad, CA, USA) and the
resulting plasmid was designated pKMUSD (Fig. 2). The allelic exchange vector pKMUSD extracted from DH5α was
introduced into the dam−dcm− E. coli host strain JM110 by electro-
2.5. Measurement of the effects of restriction enzyme I-SceI on the poration (1.8 kV, 25 μF, 200 Ω) using a Gene Pulser II electroporator
elimination kinetics of the allelic exchange vector pKMUSD (Bio-Rad, Hercules, CA, USA) to obtain demethylated plasmid DNA for
transformation into B. anthracis strain A16R. Electroporation-compe-
For plasmid elimination, B. anthracis strain A16R (pKMUSD) was tent cells of B. anthracis strain A16R were prepared as described pre-
grown in LB medium without kanamycin and A16R viously. Then, 5 μl of plasmid DNA (1 μg in water) was added to a 50-μl
(pKMUSD + pSS4332) was grown in LB medium with kanamycin at aliquot of competent cells and pulsed (2.5 kV, 25 μF, 200 Ω) in a 0.2-cm
42 °C for 12 h. Then, the samples were diluted and plated onto LB agar gap cuvette (Shatalin and Neyfakh, 2005). The cells were resuspended
with or without spectinomycin. The plates were incubated at 30 °C until in 1 ml of brain–heart infusion supplemented with 0.5% glycerol and
colonies appeared. Both strains were passaged up to four times in the incubated for 3 h at 30°C with aeration. The recovered cells were spread
same medium and the plasmid elimination rates of each generation onto LB agar plates containing spectinomycin. Spectinomycin-resistant
were analyzed as described previously (Wolfson et al., 1982). colonies were picked, and PCR was performed using the primer pair
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T. Wang et al. Journal of Biotechnology 271 (2018) 8–16
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T. Wang et al. Journal of Biotechnology 271 (2018) 8–16
pKSV7_F/R to confirm that recombinant pKMUSD had been introduced galactosidase expressed by the pclpB–bgaB gene cassette on the acceptor
into B. anthracis strain A16R. This strain was then electroporated with vector pKMBKI and the X-Gal on the plate. When the Golden Gate re-
pSS4332, which contains the gene encoding the I-SceI enzyme. The striction–ligation product was transformed into E. coli DH5α cells, al-
transformants were selected at 30 °C on LB medium containing specti- most all of the transformants were white, indicating that the three entry
nomycin and kanamycin. Fluorescent colonies carrying pSS4332 were DNA fragments had been inserted into acceptor vector pKMBKI in the
visible after 16–20 h. designed order, replacing the original pclpB–bgaB gene cassette on the
B. anthracis mutants were constructed by replacing coding se- acceptor vector. This indicated that this cloning system had high effi-
quences with the spcr cassette, which confers spectinomycin resistance. ciency and the blue/white colors enabled easy screening of the re-
Transformation and the selection of transformants in B. anthracis were combinant clones.
conducted as described previously. A single colony of A16R Twenty white colonies were picked up and analyzed by colony PCR
(pKMUSD + pSS4332) was transferred into liquid medium with kana- using three pairs of primers, eagup_F/R, eagdn_F/R and spcin_F/R. In
mycin and incubated with shaking for 8 h at 30 °C; then, the cultures these reactions, genomic DNA of B. anthracis strain A16R and plasmid
were shifted to 37 °C for 8 h. After three passages in the same medium, DNA of pSET4s were used as positive control templates to amplify the
serial dilutions of the culture were plated onto LB agar plates containing DNA fragments of eagup, eagdn and spcin. Genomic DNA of strain DH5α
spectinomycin and kanamycin, and incubated overnight at 37 °C. At was used as a negative control template in all tests. The results showed
least 50 colonies were screened for spectinomycin resistance and that all of the colonies except for No. 8 and No. 20 contained the correct
chloramphenicol sensitivity (SpcrCms). Colonies (SpcrCms) thought to eagup amplified fragment (Fig. 3A). For the spcin DNA fragment, 19 of
have undergone a double-crossover recombination event were vali- the 20 colonies contained the correct amplified DNA bands (Fig. 3B).
dated by PCR analysis using three pairs of primers, eagus_F/R, eagsd_F/ For the eagdn DNA fragment, 18 of the 20 white colonies contained the
R and eagin_F/R, and DNA sequencing. Strains with the desired gene correct amplified DNA bands (Fig. 3C). In total, 17 of the 20 colonies
replacement were passaged up to three times in the absence of anti- possessed the desired ligation of the three entry fragments eagup, spc
biotics to ensure the loss of pSS4332, which was confirmed by the loss and eagdn into the acceptor vector pKMBKI, giving a success rate of
of fluorescence. The eag gene deletion was further verified by western 85% (Fig. 3).
blot analysis using an antibody against EA1 protein. The resulting To further test the restriction–ligation efficiency, two types of re-
colony was designated A16R△eag::spc. striction–ligase buffer, namely, ligase buffer from Promega and ligase
buffer from New England Biolabs, were also tested. The amount of T4
DNA ligase used in the reaction was also tested in the range of
3. Results and discussion 1.125–2.25 units. Although increasing the amount of T4 DNA ligase
slightly enhanced the restriction–ligation efficiency, no significant dif-
3.1. Efficiency of Golden Gate restriction–ligation and identification of the ference was detected between any of the treatments, indicating a degree
recombinant allelic exchange vector of flexibility in the choice of ligase buffer and amount of ligase used in
this restriction–ligation cloning system. Engler et al. had already proved
When the acceptor vector pKMBKI was introduced into E. coli DH5α that incubation for 30 min was optimal for experiments in which three
cells, all transformants were blue because of the reaction between the β-
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T. Wang et al. Journal of Biotechnology 271 (2018) 8–16
cells. Thus, it is difficult to use the RecE/T system in this case. As such,
research has focused on increasing the allelic exchange efficiency be-
tween the chromosome and the target plasmid. Plasmids usually used in
B. anthracis based on the widely used pE194ts replicon (e.g., pMAD,
pKSV7) were able to replicate in B. anthracis, but curing was inefficient
even at the highest temperature tolerated by this bacterium (44 °C), at
which more than 50% of the cells retained the plasmid in free form. The
pWV01 replicon-based plasmids also require more than 20 passages to
lose the target plasmids, which is impractical and even then does not
guarantee success (Shatalin and Neyfakh, 2005). Thus, maintenance at
a nonpermissive temperature is critical during the passage of cells in the
absence of antibiotic selection conferred by the plasmid. The approach
to facilitating this recombination event involves the use of site-specific
Fig. 4. Kinetics of plasmid pKMUSD elimination fromBacillus anthracis strain A16R. cleavage, mediated by the restriction enzyme I-SceI, of a cognate I-SceI
B. anthracis strain A16R (pKMUSD) was grown in LB medium without kanamycin and site present in the allelic exchange vector but not elsewhere in the
A16R (pKMUSD + pSS4332) was grown in LB medium with kanamycin at 42 °C for 12 h. bacterial chromosome. This method was originally used in E. coli, and
The samples were diluted and plated onto LB agar. At each time point, the colonies were adapted for use in B. anthracis by Stibitz and coworkers (Janes and
counted and scored for the elimination of the plasmid. Plasmid elimination rate (PER)
Stibitz, 2006; Schuch et al., 2015).
was calculated based on the following equation: PER= (1 − N/N0)×100%. Note: N0,
total colonies tested; N, plasmid-containing colonies (spcr).
To address this problem, we introduced an I-SceI site into the allelic
exchange vector. After the allelic exchange vector was transformed into
B. anthracis, a second plasmid, pSS4332, which expressed endonuclease
SceI nuclease at rates of 20%–100% (Janes and Stibitz, 2006; Tischer I-SceI, was introduced to digest the first plasmid resulting in a linear
et al., 2006). Using the protocol developed in this study, the ideal allelic exchange vector during passaging. The linear allelic exchange
mutants were obtained after only three passages by screening 50 clones. vector was forced to incorporate into the chromosome by growth at
Three independent experiments indicated that approximately elevated temperatures concomitant with selection for spectinomycin as
85%–100% (43/50, 45/50, 50/50) of the spcrcms clones had eag re- a consequence of a first homologous recombination event. The loss rate
placed by the spcr cassette in the correct manner. We also deleted of the final allelic exchange vector improved significantly using this
several other B. anthracis genes, such as BA2380, atxA, mntA, qoxA, method. This method can also simulate gene targeting using linear DNA
abrB, and sigF, using this method, with a rate of allelic exchange similar fragments to some extent and increase the homologous recombination
to that in the case of eag deletion. That our method has a higher rate of efficiency greatly. This approach will also facilitate the second re-
allelic exchange of the spcrcms clones may be because we used the combination event by both selecting against bacteria in which the
double-antibiotic screen. chromosome has been damaged and stimulating recombination of the
For Gram-positive bacteria, homologous recombination, transposi- flanking homologous sequences to repair the lesion. Repair of this break
tion mutagenesis, and group II intron-based insertion mutagenesis have by a second recombination event results in loss of the integrated
been used for gene inactivation. However, the most commonly used plasmid and the desired gene replacement strains can then be obtained
method of mutagenesis is homologous recombination. This is usually a by appropriate antibiotic resistance screening (Fig. 6).
rare event, so strategies to enhance the efficiency of homologous re- In the present study, an I-SceI site, which was situated outside the
combination are vital (Janes and Stibitz, 2006; Schuch et al., 2015; two homologous genomic sequences, was introduced into the target
Shatalin and Neyfakh, 2005). Many methods have been proposed that plasmid. Recombination between the target plasmid and genomic DNA
artificially increase the efficiency of endogenous homologous re- was forcibly induced by three factors, namely, the induction of en-
combination. The success of the λ Red recombination system for re- donuclease I-SceI to initiate site-specific cleavage, a nonpermissive
combineering in E. coli provided the basis for initial experimental de- temperature, and an antibiotic. Using this strategy, eag and several
signs (Datsenko and Wanner, 2000; Tischer et al., 2006). Using other genes of B. anthracis were deleted extremely efficiently within
mycobacteriophage-encoded recombination proteins, a recombineering approximately 3 weeks with less screening work. B. anthracis, B. wei-
system for mycobacteria was developed based on the RecE/T system henstephanensis, B. mycoides, B. pseudomycoides, B. cereus, and B. thur-
(Van Kessel and Hatfull, 2007). The same protocol has also been ap- ingiensis all belong to the B. cereus group, which are found in diverse
plied in lactic acid bacteria and B. subtilis (Van Pijkeren and Britton, habitats. Although the B. cereus group is phenotypically and genetically
2012; Van Pijkeren et al., 2012; Wang et al., 2012). However, for B. heterogeneous overall, strikingly, strains of the different constitutive
anthracis, there is no effective method for transforming single-stranded species are closely related at the chromosomal level. Therefore, we
DNA into competent cells and then ensuring its stability in B. anthracis speculated that the high-efficiency allelic exchange driven by I-SceI
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T. Wang et al. Journal of Biotechnology 271 (2018) 8–16
endonuclease described here could be applied in other species of the B. Funding sources
cereus group.
The flexibility, speed and efficiency of the multiple DNA fragment This work was supported by the National Natural Science
cloning and gene replacement technique reported here should make it a Foundation of China (grant numbers 81571958, 81601739, and
valuable tool to investigate gene function in B. anthracis and other 81671979) and the State Major Science and Technology Special
species of the B. cereus group. Projects of China (grant number 2017ZX10104002).
Conflict of Interest
4. Conclusion
The authors declare no conflicts of interest.
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