Beruflich Dokumente
Kultur Dokumente
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Key Laboratory of Systems Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, 1 West Beichen Road, Chaoyang District, Beijing 100101, China
Graduate University of Chinese Academy of Sciences, Beijing 100049, China
College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
a r t i c l e
i n f o
Article history:
Received 24 August 2010
Received in revised form 8 October 2010
Accepted 9 October 2010
Available online 14 October 2010
Keywords:
Recalcitrant Bacillus
Electro-transformation
Cell-wall weakening
Cell-membrane uidity disturbance
a b s t r a c t
Bacillus amyloliquefaciens has been a major workhorse for the production of a variety of commercially
important enzymes and metabolites for the past decades. Some subspecies of this bacterium are recalcitrant to exogenous DNA, and transformation with plasmid DNA is usually less efcient, thereby limiting
the genetic manipulation of the recalcitrant species. In this work, a methodology based on electro-transformation has been developed, in which the cells were grown in a semicomplex hypertonic medium, cell
walls were weakened by adding glycine (Gly) and DL-threonine (DL-Thr), and the cell-membrane uidity
was elevated by supplementing Tween 80. After optimization of the cell-loosening recipe by response
surface methodology (RSM), the transformation efciency reached 1.13 0.34 107 cfu/lg syngeneic
pUB110 DNA in a low conductivity electroporation buffer. Moreover, by temporary heat inactivation of
the host restriction enzyme, a transformation efciency of 8.94 0.77 105 cfu/lg DNA was achieved
with xenogeneic shuttle plasmids, a 103-fold increase compared to that reported previously. The optimized protocol was also applicable to other recalcitrant B. amyloliquefaciens strains used in this study.
This work could shed light on the functional genomics and subsequent strain improvement of the recalcitrant Bacillus, which are difcult to be transformed using conventional methods.
2010 Elsevier Inc. All rights reserved.
131
Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Table 1
Bacterial strains and plasmids used in this study.
Strains or plasmids
Strains
E. coli INV110
B. amyloliquefaciens
B. amyloliquefaciens
B. amyloliquefaciens
B. amyloliquefaciens
TA1001
TA208
H
FZB42
Plasmids
pUB110
pC194
pE194
pMK4
pDG148
pHCMC02
Relevant characteristics
References or sources
Invitrogen
CGMCCa
This study
[25], BGSCb
[7], BGSC
KanR, 4548 bp
CmR, 2910 bp
EmR, 3728 bp
E. coliBacillus shuttle plasmid, rolling circle replicative, CmR
E. coliBacillus shuttle plasmid, rolling circle replicative, KanR
E. coliBacillus shuttle plasmid, theta replicative, CmR
[40],
[41],
[42],
[43],
[44],
[45],
BGSC
BGSC
BGSC
BGSC
Ciarn Condon
BGSC
TcR, tetracycline resistance; KanR, kanamycine resistance; CmR, chloramphenicol resistance; EmR, erythromycin resistance.
a
China General Microbiological Culture Collection Center.
b
Bacillus Genetic Stock Center.
1
Abbreviations used: AG, azaguanine; MSO, methionine sulfoxide; UV, ultraviolet;
DES, diethyl sulfate; PEG, polyethylene glycol; Hepes, N-(2-hydroxyethyl)-N0 -2piperazine-ethanesulfonic acid; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol;
RSM, response surface methodology; CCD, central composite design; ANOVA, analysis
of variance.
In initial screening of the optimal medium for electro-competent cell preparation, strain TA208 was grown in various hypertonic media with different nutritional ingredients and buffering
salts, including NCM [21] (17.4 g K2HPO4, 11.6 g NaCl, 5 g glucose,
5 g tryptone (Oxoid, Basingstoke, Hampshire, UK), 1 g yeast extract
(Oxoid), 0.3 g trisodium citrate, 0.05 g MgSO47H2O, and 91.1 g sorbitol in 1 L deionized water, pH 7.2), M9YE [22] (100 ml 10 M9
salts, 3 g yeast extract, 10 g casein hydrolysate (Oxoid), 2 g glucose,
2 ml 1 M MgSO4, 100 ll 1 M CaCl2, and 91.1 g sorbitol in 1 L deionized water, pH 7.2), LBSP [20] (10 g tryptone, 5 g yeast extract, 10 g
NaCl, 50 mM KH2PO4 and K2HPO4, and 91.1 g sorbitol in 1 L deionized water, pH 7.2), LBBHIS (10 g tryptone, 5 g yeast extract, 10 g
NaCl, 10 g Brain Heart Infusion (BHI; Difco, Detroit, MI, USA), and
91.1 g sorbitol in 1 L deionized water, pH 7.2), and BHIS [23]
(34 g BHI, and 91.1 g sorbitol in 1 L deionized water, pH 7.2).
Preparation of the electro-competent cells
An overnight LB culture of the Bacillus cells was diluted 100-fold
to fresh medium for preparation of the electro-competent cells.
The bacterial growth was monitored by measuring the optical density (OD) at 600 nm using a Nanodrop 2000C spectrophotometer
(Thermo Scientic, Wilmington, DE, USA). When an OD600 reading
reached 0.5, cell-wall weakening and/or cell-membrane uidity
disturbing was performed by adding Gly, DL-Thr, Amp, or Tween
80 into the culture and continued to shake for 1 h. The cell culture
was cooled on ice for 20 min, and collected by centrifugation at
4 C, 8000g for 5 min. After washing four times with ice-cold
ETM buffer (0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol),
the electro-competent cells were resuspended in 1/100 vol of the
original culture.
Development of a combined cell-wall-weakening and cell-membrane
uidity-disturbing approach using response surface methodology
Preliminary one-way experiments indicated that Gly and DL-Thr
are effective for improving transformation efciency by weakening
the cell wall, whereas Tween 80 enhances electro-competence by
disturbing the cell-membrane uidity. Effects of cell-wall-weakening and cell-membrane-disturbing combination were evaluated
using RSM. Central composite design (CCD) was used to investigate
the effects of three independent variables on the transformation
efciency (Y), Gly (X1), DL-Thr (X2), and Tween 80 concentration
(X3), which were coded at levels of 2, 1, 0, 1, and 2, respectively
(Table 2), according to
xi X i X 0 =DX;
132
Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Table 2
Independent variables involved in CCD trials shown in actual and coded levels.
Independent variables
Symbols
concentration (%)
Tween 80 concentration (%)
Coded levels
Uncoded
Coded
2
1
X1
X2
x1
x2
2
0.8
2.5
0.9
3
1.0
3.5
1.1
4
1.2
X3
x3
0.01
0.02
0.03
0.04
0.05
Electroporation
Electro-competent cells (100 ll) were mixed with column-puried plasmid DNA (100 ng) with Plasmid Mini Kit (E.Z.N.A., OMEGA
Bio-tek, Duraville, USA), and loaded into a prechilled 1-mm gap
electroporation cuvette. After a brief incubation on ice, the cell
DNA mixture was shocked by a single 2.1 kV/cm pulse generated
by BTX ECM399 electroporator (BTX, Harvard Apparatus, Holliston,
MA, USA), with the resistance and capacitance set at 150 X and
36 lF, respectively. The cells were immediately diluted into 1 ml
of corresponding recovery medium (growth medium plus 0.38 M
mannitol [15]) and shaken gently at 37 C for 3 h to allow expression of the antibiotic resistant genes, and aliquots of the dilutions
were then spread onto LB agar plates supplemented with appropriate antibiotics. Transformation efciencies were calculated by
counting the colonies on plates after incubation at 37 C for 16 h.
Some transformants were veried by plasmid extraction and
restriction enzyme digestion.
Heat inactivation of host restrictionmodication systems
Table 3
CCD matrix of three variables in coded units and the experimental and predicted
values of transformation efciency. a
Trial number
Variable
Transformation efciency
(cfu/lg of plasmid DNA)
x1
x2
x3
Observed
Predicted
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1
1
1
1
1
1
1
1
2
0
0
2
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
2
0
0
2
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
0
2
0
0
2
0
0
0
0
0
0
1.18 106
6.23 105
8.32 105
1.01 106
7.63 105
5.21 105
2.82 105
1.12 105
6.63 105
3.30 105
4.04 105
1.09 106
9.30 105
7.64 105
1.034 106
1.056 106
1.04 106
1.022 106
1.046 106
1.03 106
1.139 106
7.989 105
8.519 105
9.856 105
7.145 105
6.285 105
4.337 105
2.799 105
4.252 105
2.482 105
3.664 105
1.200 106
8.844 105
6.742 105
1.017 106
1.017 106
1.017 106
1.017 106
1.017 106
1.017 106
a
B. amyloliquefaciens TA208 was cultured aerobically in NCM with various
amounts of Gly, DL-Thr, and Tween 80 added to the medium until OD600 reached 0.5.
The competent cells were prepared and transformed with 100 ng pUB110 DNA as
demonstrated under Materials and methods.
Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
133
(data not shown). Therefore Amp was no longer used in the following RSM designs.
A positive effect of Tween 80 was also observed on strain TA208
transformation. When supplemented at a concentration of 0.03%,
transformation was elevated to 1.10 105 cfu/lg plasmid DNA
(Fig. 2d).
Combinatorial cell-wall-weakening and cell-membrane uiditydisturbing strategy with RSM design
Y is the transformation efciency, and x1, x2, and x3 are concentrations of Gly, DL-Thr, and Tween 80 in coded values, respectively.
Fig.2. Plots of transformation efciency against concentrations of the cell-wall-weakening or cell-membrane uidity-disturbing agents. Strain TA208 was cultured in NCM to
OD600 reading of 0.5. Gradient concentrations of the cell-wall-weakening or cell-membrane uidity-disturbing agents (06% of Gly (a); 02.5% of DL-Thr (b); 0100 lg/ml of
Amp (c); 01% of Tween 80 (d)) were supplemented to the culture, and the cells were shaken for 1 h before the competent cells were prepared. Data shown are averages of at
least three independent experiments with a SD.
134
Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Table 4
ANOVA of the regression model.
a
b
c
Resource
SSa
DFb
MSc
F value
Prob > F
Model
Residual
Lack of t
Pure error
Total
1.688 1012
1.891 1011
1.884 1011
7.280 108
1.878 1012
9
10
5
5
19
1.876 1011
1.891 1010
3.767 1010
1.456 108
9.92
0.0007
258.75
<0.0001
Table 5
Signicance of the regression coefcients for the response surface model.
Model term
Coefcient
DF
Standard error
F value
Prob > F
Intercept
x1
x2
x3
x1x2
x2x3
x1x3
x21
1.017 106
1.938 105
1.591 105
7.694 104
1.938 104
4.125 103
4.125 103
5.099 104
1
1
1
1
1
1
1
1
5.485 104
3.438 104
3.438 104
3.438 104
4.862 104
4.862 104
4.862 104
2.742 104
31.78
21.41
5.01
0.16
7.2 103
7.2 103
3.46
0.0002
0.0009
0.0492
0.6986
0.9341
0.9341
0.0926
x22
x23
1.126 10
2.742 10
16.86
0.0021
1.241 105
2.742 104
20.48
0.0011
135
Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Table 6
Transformation efciency of strain TA208 in various ETM buffers under optimal conditions.
Additives in ETM buffer
Concentration
Transformation efciency
(cfu/lg of plasmid DNA)
Reference
PEG 6000
Hepes
TrisHCl
KH2PO4 and K2HPO4
MgCl2
KH2PO4, K2HPO4, and MgCl2
10%
1 mM (pH
1 mM (pH
1 mM (pH
1 mM
0.25, 0.25,
[19]
[17]
[32]
[32]
[32]
[32]
7.0)
7.0)
7.0)
and 0.5 mM (pH 7.0)
Table 7
Effects of heat inactivation of host-restriction systems on transformation efciency (cfu/lg of plasmid DNA).
pMK4
Unheated
Heated
a
Syngeneic plasmids
Unmethylated
M. BamHI methylated
pC194
pE194
pHCMC02
pDG148
Not determined.
Discussion
In the current work, an electroporation protocol to transform
recalcitrant B. amyloliquefaciens has been systematically optimized,
in which the culture medium, cell-wall-weakening and cell-membrane uidity-disturbing agents, electroporation buffer, and heat
inactivation of host restriction enzyme were all investigated to
achieve a high transformation efciency. The improvement of
transformation efciency after each optimization step and the proposed electroporation method for recalcitrant B. amyloliquefaciens
are shown in Supplementary Table 1 and Supplementary Table 2,
respectively.
Culture medium is a key factor for determining the transformation efciencies of bacteria. E. coli cells grown in SOB medium usually yield higher transformation efciencies than those grown in LB
and 2 YT media [26]. In addition, the nutrient BHIS is regarded as
an appropriate medium for preparation of the competent cells in C.
glutamicum [27]. Nevertheless, we found that the nutritional ingredients in growth medium exerted a negative effect on the transformation efciency in B. amyloliquefaciens, possibly due to the high
sporulation rate of Bacillus in a rich medium [28]. Since the Bacillus
endospores are surrounded by a morphologically complex coat,
consisting of peptidoglycan, dipicolinic acid, and protein, and conferring the spores resistant to heat, UV, and other extreme environments, the spores are hardly accessible by plasmid DNA [29];
whereas the walls of vegetative cells grown in semicomplex NCM
medium are relatively loose and easy to form pore during electroporation, and subsequently more accessible by exogenous plasmid
DNA. Moreover, buffering salts in NCM, such as potassium phosphate and sodium citrate, enhance the transformation efciency
by providing the cells appropriate ions and pH.
Gly and DL-Thr have been used as cell-wall-weakening agents in
preparation of the electro-competent cells in various Gram-positive
bacteria. By replacing the L- and D-alanine, Gly and DL-Thr can integrate into the tetrapeptide, which is the linker of N-acetylmuramic
acid in the cell wall, reduce the cross-linking of the peptidoglycan
layer, and thereby make the cell wall more accessible by exogenous
DNA [30]. Lactococcus lactis [31], B. cereus [32], and C. glutamicum
[33] grown in Gly-rich medium were reported to show a higher
transformation efciency than those grown in Gly-poor medium,
and DL-Thr enhanced the transformation efciency of B. subtilis
[30] and Pediococcus acidilactici [34]. In this study, we also found that
Gly and DL-Thr loosen the cell wall of B. amyloliquefaciens and improve the transformation efciency. Similar observations have been
136
Electroporation of recalcitrant B. amyloliquefaciens / G.-q. Zhang et al. / Anal. Biochem. 409 (2011) 130137
Acknowledgments
We are grateful to BGSC and Prof. Ciarn Condon for providing
the plasmids and strains used in this study. We thank Prof. Yongsheng Che for English revision. This research was funded by the
National Drug Discovery Program of China (2008ZX09401-05),
and the National Key Program of Research and Development of
China (2008BAI63B01).
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