A continuous process to extract plasmid DNA based on alkaline lysis

Abstract

Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants and then ethanol precipitated. This process replaces all the centrifugation steps before obtaining crude plasmid and can be easily scaled up to meet demands for larger quantities. Using this procedure, plasmid can be extracted and purified from 4 l of Escherichia coli culture at an OD 600 nm of 50 in <90 min. The plasmid yields are approximately 80-90 mg l(-1) culture.

Full text

A continuous process to extract plasmid DNA based on
alkaline ly sis
Xiaolin Li
1
, Huali Jin
1
, Zhifang Wu
1
, Simon Rayner
1,2
& Bin Wang
1
1
State Key Laboratory for Agro-Biotechnology and the Key Laboratory of Agro-Microbial Resources and Applications of MOA, China Agricultural University, Beijing
100094, China.
2
Present address: Wuhan Institute of Virology, Chinese Academy of Science,Wuhan, Hubei 430072, China. Correspondence should be addressed to B.W.
(bwang3@cau.edu.cn).
Published online 17 January 2008; doi:10.1038/nprot.2007.526
Rapid advances in the fields of DNA vaccines and gene therapy have produced increased demands for large quantities of recombinant
plasmid DNA. The protocol presented here extracts plasmid DNA in a scalable continuous process based on an alkaline lysis protocol.
In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to effect lysis and control alkalinity.
The resulting solution is passed through a series of filters to remove contaminants and then ethanol precipitated. This process
replaces all the centrifugation steps before obtaining crude plasmid and can be easily scaled up to meet demands for larger
quantities. Using this procedure, plasmid can be extracted and purified from 4 l of Escherichia coli culture at an OD 600 nm of 50 in
o90 min. The plasmid yields are B80–90 mg l
1
culture.
INTRODUCTION
Rapid advances in the fields of DNA vaccines and gene therapy
1,2
have produced increased demands for large quantities of recombi-
nant plasmid DNA. The problem is further exacerbated, as efficiency
of plasmid-based delivery to cells is relatively low—only 0.1% of
plasmids presented to the cells can reach the nucleus and be
expressed
3–5
. Thus, a scaleable process for the continuous prepara-
tion of plasmid DNAwould be of great benefit to researchers and is
a problem that has been the focus of several previous studies
5,6
.
Broadly speaking, a process for manufacturing plasmid DNA
includes the following steps: plasmid construction, cell transforma-
tion, cell growth, and extraction and purification of the plasmid
4,7
.
For extraction and purification, the most critical part of the process
is probably cell breakage
8
and there are several ways to achieve this.
The most popular method is based on alkaline lysis of the cell
9
.The
principal drawback with this method, with a view to automating
the process, is that it includes multiple centrifugation steps. The
first step separates the bacteria from the cultured media before lysis,
the second step separates the plasmid and RNA from the cell debris
after lysis, and the third step separates the precipitated plasmid
from above solution after ethanol or isopropanol precipitation
10,11
.
These steps cannot be easily incorporated into a continuous process
and so impede scaling up the alkaline lysis method for large-scale
preparation of plasmid DNA
12
. An alternative method for cell
breakage is boiling lysis
13
; however, in this approach, the yield and
purity of the plasmid is inconsistent and the complexities of the
method make it undesirable even for research-scale preparation
14
.
Recently, we reported the successful development and optimization
of a continuous process based on the thermal lysis protocol
15,16
.
The process was modified from the conventional boiling lysis
protocol and made plasmids in a continuous and scalable manner.
However, a centrifugation step was still required after lysis.
Large-scale extraction of plasmid by the alkaline lysis procedure
has been recognized as problematic, specifically due to the three
separate steps involved in the sequential mixing and reaction of
each of the three solutions (solutions I, II and III as indicated in
REAGENT SETUP) used in the process. In a more recent report
17
,
we described a design for a flow-through protocol from cultured
bacteria for the extraction of plasmid DNA by the alkaline lysis
procedure, with the incorporations of additional modifications to
permit a more continuous process.
The schematic design of the device and setup is shown in
Figure 1. One of the most important modifications to the process
was the elimination of the centrifugation steps, as these represent
the most rate-limiting segment for plasmid extraction using the
alkaline lysis method. In addition, we examined the various process
parameters to improve the overall efficiency and the quality and
yield of final product. The modular design of the system ensures
that the operation can be easily scaled up and experimental
parameters easily reinvestigated to ensure optimum operation for
alternative configurations.
To eliminate centrifugations, we employed a 0.2-mm hollow fiber
cartridge to harvest cells, a filtration system (labeled N in Fig. 1)to
remove debris after cell lysis, and a 70-mm nylon filter to remove
RNA contamination after the plasmid was precipitated in ethanol.
As the hollow fiber cartridge can be manufactured in many
different sizes, a suitable cartridge can be selected to recover the
desired final volume of fermented bacteria. After mixing with
solution III, lysed bacteria are passed through four layers of
nylon filters of gradually decreasing pore size (375, 186, 122 and
70 mm) to remove debris and other impurities in this size range.
The use of a 70-mm nylon filter has been observed to be the most
effective at separation of RNA from the plasmid after ethanol
precipitation.
In the process of preparation of plasmid, the most critical step is
probably cell breakage
9
. If the breakage step is too vigorous, the
genomic DNA will be broken up and contaminate the plasmid. If
the step is too gentle, less plasmid DNA will be obtained and overall
yield will fall. In this protocol, the incorporation of mixing chamber A
(labeled A in Fig. 1) addresses both these issues and permits
efficient lysing of the bacteria. By investigating mixing order of
reagents and flow rate, we were able to determine the best
combination in terms of optimal yield and quality of final product.
An additional problem was that, after mixing with lysing
solution II in chamber A, the lysed product was very viscous and
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difficult to mix with solution III. This
mixing step is also an important part in
the process because the alkalinity of the
lysed product must be reduced rapidly to
avoid secondary reactions that can
adversely affect the quality of the final
product. We overcame this problem by
including a second mixing chamber B
(labeled B in Fig. 1), combined with a
shaker, to assist in the mixing process. By
examining a range of frequencies for the
shaker, we were able to determine which
produced the best results for the final
product in terms of quality and yield.
The procedure provides a fast and effi-
cient way to extract plasmid in large
volumes. After extracting plasmid using
the described protocol and continuous
device, the plasmid may be purified using
any available purification protocol. We have
compared the extracted product obtained
from our system with the product obtained
from a manual alkaline lysis. Both were
purified in Qiagen plasmid-purification
columns (Qiagen, Germany) and compar-
able transfection efficiency was observed in
the samples.
In summary, we have developed a novel
design comprising continuous control of
reagent flow into the system and replace-
ment of all the centrifugation steps by use
of filters and hollow fiber membrane car-
tridges. The resulting continuous process
provides a scalable, faster and more effi-
cient method for plasmid extraction, which
is readily adoptable for various subsequent
downstream purification protocols such as
spermidine compaction
18
, CTAB precipita-
tion
19,20
, gel filtration
12
or magnetic bead
purification
21
. The process circumvents
problems of traditional alkaline lysis and
provides a platform for automated produc-
tion of plasmid DNA to meet the needs of
DNA vaccinations, non-viral vector-based
gene therapy, RNAi expressing constructs
and other related clinical and research
applications. Although this continuous sys-
tem is easy to operate, an initial step invol-
ving adjustment of connections, tube sizes
and synchronizing pumps is required to set up and optimize the
system. Flow rate, the size of mixing cells, and shake frequency need
to be optimized for new experimental conditions. The fermenta-
tion process and harvesting of bacteria are not described in the
following procedure, which begins with the alkaline lysis of
harvested bacteria. Fermentation should be carried out as described
in ref. 15, and bacteria should be harvested for use in this protocol
as described in ref. 15, using Solution I instead of TE.
MATERIALS
REAGENTS
.
Plasmid DNA m CRITICAL The following plasmids have been extracted using
this protocol: pcDNA3(5446 bp), pGEX-4T-1(4969 bp), pVAX1(2999 bp)
and pcD-VP1. The last, encoding a capsid protein VP1 (639 bp) of foot and
mouth disease virus (described previously
22
), was extensively used during the
optimization of this protocol.
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Concentrated
bacteria
T1 T2
C1
P1
T5
P1 P1P1
C2
C3 C4
T3 T4
A
B
N
Shaking
Precipitate
70% EtOH
wash
Air dry
Nylon
fiter
C5 C6
100%
Ethanol
Figure 1
|
Schematic of continuous alkaline lysis process to extract plasmid DNA from Escherichia coli.The
cells in C1 are adjusted to an OD 600 nm of 100 with solution I. P1 (4-channel peristaltic pump) is turned
on in order to pump the diluted bacteria, 0.4 M NaOH, 2% SDS, and solution III from containers C1, C2, C3
and C4, into mixing chamber A, through T1, T2, T3 and T4 tubes, respectively. The speed of the pump is
adjusted to give a flow rate of 3.0 cm s
1
in tubes. During the mixing, 0.4 M NaOH and 2% SDS meet first at
the junction of tubes T2 and T3 and are mixed to form solution II. Solution II then mixes with the diluted
bacteria in mixing chamber A to cause lysing of the bacteria. Lysed bacteria mix with solution III in mixing
chamber B, which is facilitated by the shaker (at a frequency of 75 Hz). Most proteins and chromosomal
DNAs form white precipitates and are removed by passing the liquid through filter N comprising four
sequential nylon filters of 375, 186, 122, 70 mm pore sizes. The filtered liquid is collected in container C5
and the total collected volume is measured. Plasmid DNA in the liquid is precipitated by adding two times
its volume of 100% ethanol into chamber C5 and is collected on a nylon filter of 70-mm pore size. The
precipitated plasmid DNA on the filter is rinsed using 70% ethanol in 2 liquid volume above (waste liquid
is collected in container C6), air dried and resuspended into TE buffer for further purification.
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.
Escherichia coli:DH5a (Invitrogen) m CRITICAL TOP10 and JM109 can also
be used.
.
Tris–base (Sigma, cat. no. T1819)
.
EDTA (Sigma, cat. no. T3913)
.
Glucose (Beijing Chemical Factory, cat. no. 3475-1999)
.
0.4 M NaOH (Beijing Chemical Reagents Company, cat. no. 10019792)
m CRITICAL Use freshly made.
.
2% (wt/vol) SDS (Sigma, cat. no. L5750)
.
Potassium acetate (Beijing Chemical Reagents Company,
cat. no. 30154592)
.
Acetic acid (Beijing Chemical Reagents Company, cat. no. 10000292)
.
70% Ethanol (Beijing Chemical Factory, cat. no. 32061)
.
10 mg ml
–1
Ethidium bromide (EtBr; Sigma, cat. no. E7637)
.
Agarose (Biowest, cat. no. A6585)
.
DNA markers (TaKaRa, cat. no. D501A)
EQUIPMENT
.
4-Channel peristaltic pump (Hitachi Density Gradient pump, type: DGP-2.
Hitachi Koki)
.
Multi-Wrist Shaker (LAB-LINE Instruments)
.
Filter assembly (see EQUIPMENT SETUP)
.
Mixing chambers A and B (see EQUIPMENT SETUP)
.
Containers (see EQUIPMENT SETUP)
.
Tubing to connect the components of the apparatus (see EQUIPMENT
SETUP)
REAGENT SETUP
TE buffer 10 mM Tris–HCl, 1 mM EDTA, pH 8.0. Can be stored at room
temperature for one year.
Solution I 50 mM Glucose, 25 mM Tris–HCl, 10 mM EDTA, and pH 8.0. Can
be stored at 4 1Cfor6months.
Solution II Mixture of 0.4 M NaOH and 2% SDS in equal volumes (see Fig. 1).
Solution III 3 M potassium acetate, 5 M acetic acid, and pH 4.8. Can be stored
at 4 1Cfor6months.
TAE buffer 40 mM Tris–acetate, 1 mM EDTA. Can be stored at room
temperature for one year.
EQUIPMENT SETUP
Filter assembly Nylon filters with pore sizes of 375, 186, 122 and 70 mm
(Wangyi, cat. no. D501A) are stacked sequentially in a cassette type assembly, with
the largest pore filter at the front of the device (i.e., receiving the solution first) and
thesmallestporefilterattheend.Filtersshouldbediscardedaftersingleuse.
Mixing chambers A and B The glass chambers are of circular cross-section
(2-cm diameter) and elliptically shaped along the longitudinal axis (8-cm long),
(e.g., similar in shape to a rugby ball or American football). The chambers were
constructed in house and are of 18.1 ml volume.
Containers Containers C1, C2, C3, C4, C5 and C6 are 25 cm in diameter and
20 cm in depth (can be obtained from a hardware store).
Tubing to connect the components of the apparatus Silicone tubing with an
inner diameter of 3.10 mm (Tygon, R-3603) is used for the connection. The
length of each tube is: T1, 15.3 cm; T2 and T3, 14.7 cm; T4, 20.9 cm; T5, 6.2 cm.
PROCEDURE
Continuous alkaline lysis preparation

TIMING 45 min
1| Dilute bacteria that were fermented and harvested as described in ref. 15 (using solution I instead of TE for the harvesting
procedure) with solution I (in container C1 of Fig. 1) until the OD value at 600 nm reaches 100. The typical starting volume is 250 ml.
m CRITICAL STEP The solution of bacteria in solution I should be well mixed.
m CRITICAL STEP 100 OD 600 nm is the optimal density for the lysis. A lower density will cause excessive lysis, leading to secondary
products, and a higher density will result in incomplete lysis
17
.
2| Turn on P1 pump to pump diluted bacteria, 0.4 M NaOH, 2% SDS, and solution III from containers C1, C2, C3 and C4, into
mixing chambers A and then B, through tubes T1, T2, T3 and T4, respectively (see Fig. 1). Adjust the speed of pump, and make
the flow rate 3.0 cm s
1
for each solution.
m CRITICAL STEP A lower or higher flow rate can result in lower plasmid yield
17
. The 0.4 M NaOH and 2% SDS meet first at the
junction of tubes T2 and T3, and mix to produce solution II. Solution II then mixes with diluted bacteria in mixing chamber A and
lyses the bacteria. Lysed bacteria mix with solution III in mixing chamber B and the shaker (at a frequency of 75 Hz) ensures
complete mixing.
m CRITICAL STEP 75 Hz is the optimal shaker frequency; a lower frequency will cause incomplete lysis and a higher frequency can
result in more chromosomal DNA contamination
17
.
3| The proteins and chromosomal DNA after the mixing of solution III form white precipitates, which are removed by
passing continuously through T5 onto the filter N comprising four sequential nylon membranes of progressively smaller pore size
(375, 186, 122 and 70 mm) and the filtered liquid is collected in container C5 (see Fig. 1).
m CRITICAL STEP Nylon membranes of the stated pore sizes (from bigger pore to smaller pore size) filter the mixture and will make
this step quicker and more efficient. Nylon membranes of smaller pore sizes may also work well, but this has not yet been
investigated thoroughly.
4| Measure the volume in C5 and add cold 100% ethanol at 2 volume of the collected liquid to precipitate plasmid DNA.
m CRITICAL STEP Store the ethanol at 20 1C for 2 h before using. If the volume of bacterial culture in C1 is known to be the same in
subsequent fermentation batches, the same volume of 100% ethanol as used in this step can be pre-added in C5 for subsequent
processes.
5| Collect precipitated plasmid DNA by passing solution through a nylon filter of 70-mmporesize.
m CRITICAL STEP The precipitated plasmid DNA remains on the filter.
6| Rinse precipitated plasmid DNA on the nylon filter with 70% ethanol at 2 volumes of the collected liquid in C5.
m CRITICAL STEP Rinses with 70% ethanol are critical to remove excess RNA contamination; more than two rinses can be applied.
Plasmid DNA purification

TIMING 30–40 min
7| Remove precipitated plasmid DNA from the filter and resuspend in 1 ml of TE buffer for further purification. Use a commercially
available purification column to purify plasmid according to the manufacturer’s instructions, such as the Qiagen-tip available from Qiagen.
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Plasmid DNA quantification
8| Serially dilute plasmid DNA (from 100 to 15,000-fold) and
run 5 ml diluted plasmid DNA mixed with 1 mlloadingdyeona
0.7% agarose gel (at 100 V for 45 min) along with a standard
quantitative DNA marker. Visualize bands under 300 nm UV
light after staining with EtBr and record with suitable digital
camera (e.g., Olympus). Quantify each band in comparison to
the intensity of a known amount of DNA marker using the
Quantity One software package (Bio-Rad).
m CRITICAL STEP DNA Markers or ladders from other companies
can be used in this method if they have bands that are serially
quantitated.
Plasmid quality analysis
9| Plasmid quality can also be analyzed by measuring its ratio of OD 260/280 nm using UV-visible spectrophotometry. The
ratio of the OD values at 260 nm versus those at 280 nm should be 1.8–2.0, which is within the range of acceptable purity for
plasmids. The contaminants, such as bacteria proteins can be visualized by silver stained gels and adapted from the Bio-Rad
Silver Stain Kit Protocol (Bio-Rad). The level of lipopolysaccharides or endotoxin can be tested by the Limulus Amebocyte
Lysate gel-clot assay (Associates of Cape Cod).

TIMING
Steps 1–6, continuous alkaline lysis and plasmid precipitation: 45 min
Step 7, plasmid DNA purification: 30–40 min
Step 8, plasmid DNA quantification: 1 h
Step 9, analysis of plasmid DNA quality: 10 min
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
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MA
OC
SC
TABLE 1
|
Troubleshooting table.
Problem Possible cause Solution
Low plasmid yield Cell concentration is too low Optimize the fermentation conditions
Cell concentration is too high Adjust the cell concentration to 100 OD 600 nm in Step 1
Excessive lysis occurred owing to
low flow rate
Flow rate should be optimized for new experimental conditions
Wrong mixing order Make sure the connectivity of tubes will allow 0.4 M NaOH and
2% SDS to mix first, then with bacteria
Low shaking frequency Adjust the shaking frequency of mixing cell B to 75 Hz or
reoptimize if experimental configuration has been changed
Excessive ghost band
24
in front
of supercoiled plasmid DNA
Cell concentration too low Adjust the cell concentration to 100 OD 600 nm as in Step 1
Change to different strains of Escherichia coli
Flow rate is too slow Adjust the pump speed to increase the flow rate to B3.0 cm
1
Mixing cell A is too small Make mixing cell A bigger (to same proportion if using
wider tubes)
Excessive amounts of
contaminated
chromosomal DNA
Cell concentration too high or too low Adjust the cell concentration to 100 OD 600 nm as in Step 1
The pH may have changed in one or more
of the solutions
Use freshly made solutions I, II and III
The shaking frequency is too high in cell B Readjust the shaking frequency in cell B
Figure 2
|
Electrophoresis of plasmid
extracted from the continuous alkaline lysis.
The plasmid was extracted by manual alkaline
lysis
13
and by the continuous alkaline lysis
described by this protocol. After
purification with the previously described
protocols
23
,1ml plasmid was subjected to
electrophoresis on a 0.7% agarose gel to
determine its purity and yield. M, extracted by
manual alkaline lysis; A, extracted by
continuous alkaline lysis. SC, plasmid in
supercoiled form; OC, plasmid in open circle
form; genomic DNA contamination is shown as a
band above the OC.
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ANTICIPATED RESULTS
Plasmid yields and quantities from four independent trials are
shown in Table 2. In general, this protocol offers a procedure to
extract plasmid in a continuous method and eliminates all the
centrifugation steps before plasmid purification. The plasmid
yield after purification is 80–90 mg l
1
and can be easily scaled
up by using bigger tubes and pumps. After purification, the ratio
of plasmid is B1.8–1.9 (Table 2), and most DNA are supercoiled
(Fig. 2).
Published online at http://www.natureprotocols.com
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reprintsandpermissions
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TABLE 2
|
Plasmid yield from this protocol.
Plasmids
a
Escherichia coli
b
Yield (mg l
1
) OD (260/280)
pcDNA3 DH5a 90.16 1.89
pVAX1 DH5a 80.00 1.85
pcDNA3-VP1 JM109 85.47 1.82
pEGFP-N3 TOP10 83.33 1.88
a
pcDNA3.1, pVAX1 and pEGFP-N3 are plasmid vectors obtained from Invitrogen. pcDNA3-VP1 was
obtained by cloning of the VP1 protein of foot and mouth disease virus.
b
All plasmid DNA were
transformed into E. coli and fermented in the semi-synthetic medium in an automatic fermenter as
described in ref. 15.
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