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USE OF FACTORIAL DESIGN AS AN

APPLICATION OF THE OPTIMIZATION OF
EXPRESSION OF TAQ DNA POLYMERASE I
CLONED IN E. COLI

Article · December 2013

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 Annals of West University of Timisoara
S e r i e s C h e m i s t r y 2 2 ( 3 - 4 ) ( 2 0 1 3 ) 67 - 8 0

USE OF FACTORIAL DESIGN AS AN APPLICATION

OF THE OPTIMIZATION OF EXPRESSION OF TAQ
D N A P O L Y M E R A S E I C L O N E D I N E. C O L I

Gh e or g hi ţa M e n g hi u a , b , M . C u ţi tar b , Mar ija Bla zi c c , B e atr ic e Vl ad - Or o s a , b ,

V. O s taf e a , b
a
Advanced Research Environmental Laboratories, Multidisciplinary Research
Platform “Nicholas Georgescu - Roegen”, 4 Street Oituz, Timisoara 300086,
Romania
b
Department of Biology – Chemistry, Faculty of Chemistry – Biology – Geography,
West University of Timisoara, 16 Street Pestalozzi, Timisoara 300115, Romania
c
Department of Biochemistry, Faculty of Chemistry, University of Belgrade,
Studentski trg 12, 11000 Belgrade, Serbia

Received: 2 December 2013 Modified 5 December 2013 Accepted 17 December 2013

SUMMARY

The optimization of the experiments can help the researcher reduce

the number of experiments, revel the factors that significantly influence the
response variable and emphasize the possible interactions between the
independent variable of the experimental model. As an application of a 32 full
factorial design, the optimization of the protocol used for purification of a
plasmid (pET22) containing the gene of Taq DNA polymerase I was performed.
For a 23 full factorial design, the application was the expression of the gene
(Taq DNA polymerase I cloned in pET22 plasmid) by E. coli BL21 strain.
Key words: design of experiments; DOE; factorial design; Taq DNA Polymerase;
gene expression;

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 M E N G H I U G . , C U T I TA R M . , E T A L .

INTRODUCTION

It is a well-established practice for the researchers to share their bacterial

strains, cloned genes, plasmids or other materials used in the molecular biology
laboratories. Sometimes, trying to reproduce some published results, following the
receipts of the authors, it happens to not lead to the same or similar results and in these
circumstances the first thought is to consider that the cause of the failure is either the bad
quality of the biological material received as a gift or the published procedure used by
the authors. In such cases, a better approach is to try to adapt the published protocols to
the actual conditions in your own laboratory, i.e. to make an optimization of the entire
procedure based on the biological material you have received.
As we faced such situation with E. coli cells containing a plasmid that encoded
Taq DNA polymerase [1], after several failures, we have decided to design some
experiments, first to prove that the bacterial cells indeed contains the published plasmid
and gene and further to optimize the steps required to express the gene on interest. As
such goal may be cumbersome, tedious and resource consuming, an approach to design
the experiments by factorial design procedure was applied. More exactly, in the present
paper we describe a simple procedure to verify if the starting material, i.e. the cells of E.
coli (strain BL21) contain the plasmid (pET22 b(+)) with the gene coding the Taq DNA
polymerase I and a simple optimization procedure of the process of expression of this
gene. The design of these experiments was performed using Minitab software (Minitab
Inc., State College, PA, USA).

MATERIALS AND METHODS

Reagents. All reagents were analytical or molecular biology grade: yeast extract
(Carl Roth, #2363.3), agar (Carl Roth, #X928.1), casein hydrolysate (Carl Roth,
#A157.1), NaCl (Carl Roth, #9265.1), glucose (Carl Roth, #HN06.4), EDTA
(Ethylenediamine tetra acetic acid)(Carl Roth, #CN06.3), TRIS-HCl (Carl Roth,
#4855.5), sodium hydroxide (Carl Roth, #6771.3), sodium dodecyl sulfate (SDS) (Carl
Roth, #2326.2), ethanol (Carl Roth, #P075.3), 2-propanol (isopropanol) (Carl Roth, #
AE73.2), IPTG (Isopropyl-β-D-thiogalactopyranoside) (Carl Roth, # CN08.2),
Ampicillin sodium salt (Carl Roth, #K029.2), Commercial Taq DNA polymerase
(Thermo Fisher Scientific, #EP0401)

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 FACTORIAL DESIGN APPLIED IN MOLECULAR BIOLOGY EXPERIMENTS

Solutions. Alkaline lysis solution I (GTE) [2]: 50 mM glucose, 25 mM TRIS-

HCl, 10 mM EDTA, pH 8.0. Alkaline lysis solution II (P2): 200 mM sodium hydroxide,
1% SDS. Alkaline lysis solution III (K-acetate solution): 4 M CH3COOK,
Cells and plasmid. The E. coli BL21 strain containing Taq Polymerase gene
(wtTaq) cloned in pET22 b(+) plasmid, used for expression of Taq DNA Polymerase I
experiments was ordered as a synthetic gene from Gene Script.
The expression plasmid pCTcon2 with B11GOx mutant gene insert used for Taq
DNA polymerase Activity Assay was kindly provided by Prof. R. Prodanovic from U.
Belgrade (Serbia) [3].
Methods
Organism and growth conditions [4]. From the glycerol stock of E. coli BL21
cells (stored at -20⁰C) that contained the Taq DNA polymerase I gene cloned in pET22
b(+) expression plasmid, an aliquot of 1 µL was thawed on ice and spread on a LB Amp
agar plate (with 100 µg/mL ampicillin). After 24 h at 37⁰C, one colony was used to
inoculate 1.5 mL LB Amp liquid media (with 100 µg/mL ampicillin) contained in a 2 mL
Eppedorf tube. These tubes were incubated overnight at 37⁰C and shacked at 250 rpm.
Extraction and purification of plasmid containing the cloned gene
(Miniprep)[5]. The cells cultivated as described in Organism and growth conditions
section were centrifuged at 13000 rpm for 1 min. The supernatant was removed and the
cells were resuspended in 150 µL Alkaline lysis solution I (GTE). After vigorous mixing,
300 µL Alkaline lysis solution II (P2) was added and mixed slowly by inverting tubes
until content become clear. Different quantities of Alkaline lysis solution III (K-acetate
solution) (350 µL, 450 µL, 550 µL) were added and mixed slowly by inverting tubes,
taking care that the white precipitate (most of the membrane material and genomic DNA)
do not break. The suspension from tubes was centrifuged at 13000 rpm for 4 min. After
centrifugation 0.8 mL supernatant were transferred to other tubes and different quantities
of cold isopropanol (300 µL, 400 µL, 500 µL) were added. The tubes were mixed well
and centrifuged 4 min at 13000 rpm and the supernatant was carefully discarded. The
precipitate, containing the plasmids DNA, was resuspended in 1 mL of 75% ethanol; the
tubes were inverted several times and centrifuged at 13000 rpm for 2 min. The
supernatant was removed and the open tubes were incubated for 30 min at 45⁰C in order
to dry the DNA plasmid material.

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 M E N G H I U G . , C U T I TA R M . , E T A L .

Plasmid restriction [6]. The dried plasmids obtained as described in the

previous section were gently dissolved in 20 µL of sterilized distilled water and DNA
concentration determined. A restriction mixture containing 5 µL sterilized distilled water,
4 µL plasmid solution, 0.5 µL BamHI restriction enzyme and 1 µL BamHI buffer was
prepared in PCR tubes. The reaction was incubated for 2.5 h at 37°C and stopped by
incubation of the mixture at 80°C for 20 min. Around 5 µL from these solutions were
used for electrophoresis in agarose gels.
Plasmid electrophoresis in agarose gels [7]. For electrophoresis, 5 µL
restriction mixture of each samples were mixed with 1 µL loading dye and loaded in 0.8
% agarose gel [8]. The electrophoresis was performed at 80 V, 200 mA and 300 W. When
the dye reached 2/3 of gel (45 min) the gel was colored with ethidium bromide (at a final
concentration of 0.5 µg/mL) for 10 min and photographed under UV light.
Gene expression for enzyme production [9]. For enzyme production, a colony
of E. coli BL21 that contained the Taq DNA polymerase I, that were grown in LB solid
media supplemented with 100 µg/mL ampicillin, as described in Organism and growth
conditions section, was inoculated into 3 mL LB liquid media supplemented with
ampicilin (100 µg/mL), contained in 15 mL Falcon tubes and cultivated overnight at
37⁰C under constant shaking at 250 rpm. From these tubes, portions of 0.5 mL were
mixed in Erlenmeyer flasks with 15 mL LB liquid media containing ampicillin (100
µg/mL). Growth of the cells was stopped when the optical density at 620 nm reached 0.4,
or 0.8 absorbance units, respectively, by storing the Erlenmeyer flasks at 4°C. The
expression of the recombinant gene was induced by adding isopropyl b-D-1
thiogalactopyranoside (IPTG) until 0.2 mM final concentration. The cultures were
cultivated at 37C, for 12 - 16 h under constant shaking at 100 rpm.
Purification of Taq Polymerase [10]. After cultivation of the E. coli cells as
described in the previous section for 12 or 16 h respectively, 1 mL from each induced
culture was dispensed into an Eppendorf tube and the cells were harvested by
centrifugation for 1 min at 15000 rpm. The cells were resuspended in 200 µL TEN buffer
(1 mM EDTA, 100 mM NaCl in 10 mM Tris-HCl at pH 7.9). The samples were heated at
75 or 85C in water bath for 30 min (with brief vortex mixing every 10 min). Denatured
protein and cellular debris were removed from the lysate by centrifugation at room
temperature for 10 min at 15000 rpm. The supernatant containing Taq DNA polymerase

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 FACTORIAL DESIGN APPLIED IN MOLECULAR BIOLOGY EXPERIMENTS

was recovered to a new Eppendorf tube and assayed for enzyme activity in PCR
protocols (or preserved for few hours at 4°C if needed).
Taq DNA polymerase Activity Assay [11]. Taq DNA polymerase activity assay
was made using a PCR protocol. Reaction mixture of PCR containing 35.5 µL sterilized
distilled water, 5 µL of 10xTaq polymerase buffer, 4 µL of 25 mM MgCl 2, 1 µL of 10
mM dNTPs, 1 µL of 25 µM forward primer, 1 µL of 25 µM reverse primer, 1 µL of 1
µg/mL plasmid (pCTcon2 containing B11GOx mutant gene from Aspergillus niger) and
1-2 µL Taq DNA polymerase preparation obtained in the previous section or a
commercial enzyme for positive control. For negative control, the polymerase solution
was changed with the same volume of distilled water.

RESULTS AND DISCUSSIONS

The aim of this paper was to find a mean to easily and rapidly check out if a
plasmid contained the gene of interest and further to use a procedure to prove that the
inserted gene can be expressed and the expression product is active. For all these
techniques there are published protocols but when the receipts are transferred to other
laboratories the results can be altered by some factors that can have different values in
different laboratories and their influence was not considered at the beginning of the
experiments. Sometime a slightly difference in the time of initiation of the next step, or
the omission of a step, or the practice of using other concentrations or other sources of
chemicals, in a many steps procedure can conduct to totally different results. In such
cases, it will be necessarily to assess the influence of some steps, or some factors, on the
yield of the final product that means the optimization of the entire procedure.
In many cases, the factors that influence the procedure under study can have
three or more levels (values of the independent variables). For example, the procedure
known as miniprep, used to isolate the plasmid from the bacterial cells containing the
plasmid with the gene of interest can be divided in the following steps: washing the cells
in a isotonic buffer, the lysis of the cells with a detersive (SDS) in alkaline media,
precipitation of the proteins and other cellular components from the supernatant
containing the plasmid and, finally, the precipitation of the plasmidial DNA with

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 M E N G H I U G . , C U T I TA R M . , E T A L .

isopropanol. This procedure was used to verify if the plasmid contains the gene of Taq
Polymerase I.
All these steps represent or contain factors that may influence the amount of
plasmidial-DNA produced, which is the response, or dependent variable. As in this
process there are several factors involved (independent variables) an optimization
procedure may be required when some of the factors are changed. The traditional way of
optimization is one-of-the-time procedure, when only one factor is varied during a series
of experiments, all other factors being fixed at a certain value. Then in the next series of
experiments, the other factor is changed while the others are fixed. This approach,
besides being time and resources consuming, do not necessarily conducts to the optimal
conditions for the entire procedure and it will not revels the interactions between factors.
The other approach is the design of the experiments (DOE), when all the factors are
varied in the same series of experiments and when the interactions between these factors
are sought.
For the above procedure some steps were selected as factors (independent
variables), each with three levels (32 factorial designs that requires 9 experiments). For
example, factor A was considered the concentration of potassium acetate used for the
precipitation of the proteins and other cellular components (levels 350, 450 and 550 µL 4
M solution added to the test tubes); factor B was isopropanol (levels 300, 400 and 500
µL).

Table I. The matrix design and the decoded factors for a 3 2

factorial design
Runs Matrix design Decoded factors
A B AcK IsoP
1 -1 -1 350 300
2 0 -1 450 300
3 +1 -1 550 300
4 -1 0 350 400
5 0 0 450 400
6 +1 0 550 400
7 -1 +1 350 500
8 0 +1 450 500
9 +1 +1 550 500

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 FACTORIAL DESIGN APPLIED IN MOLECULAR BIOLOGY EXPERIMENTS

A look to the main effect plots (Figure 1) of the factors considered in this series
of experiments can reveals that the increase of the isopropanol volume augments the
concentration of the plasmidial DNA (the dependent variable in this DOE). Regarding
the influence of the concentration of potassium acetate it seems that an optimal values is
450 µL. The analysis of variance reveals that the factors that are significant (based on the
fact that p is lower than 0.05) are AcK, IsoP and AcK*IsoP. This may suggests that there
is an interaction between the factors AcK and IsoP, although they belong to different
steps of the procedure.

Figure 1. The main effects plot from a 32 factorial design used for estimation of plasmid production by
E. coli BL21 strain.
From Figure 2 one can draw the conclusion that there is an interaction effect
between the precipitation of the proteins and the precipitation of the plasmidial DNA
step, but some other factors seems to be involved as the plots do not follow a pattern.

Figure 2. The interaction

plots of the 32 factorial
design used for estimation of
plasmid production by E. coli
BL21 strain

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 M E N G H I U G . , C U T I TA R M . , E T A L .

The fact that the plasmidial DNA contains the gene and the plasmid of interest
is proven by agarose gel electrophoresis. In Figure 2 the line 1 is a sample obtained by
using as restriction enzyme BamH I while in line 2 Xba I was used. In both cases the
mass of the DNA corresponds to the sum of masses of the plasmid and gene of interest.

Figure 3. The agarose-gel electrophoresis of plasmidial DNA.

The mass - ladder shows the fact that the bands in lines 1 and
2 have a mass corresponding to the sum of masses of plasmid
pET22 plus the Taq Polymerase I gene (i. e. 5,5 kb plus 2.5
kb). In line 1 the restriction enzyme used for digestion was
BamH I and in line 2 the enzyme was Xba I.

The second series of experiments have studied the influence of some factors
from the procedure applied to express the Taq Polymerase gene in E. coli. Starting from
bacterial cells caring plasmids that contains the gene of interest, for the expression of the
enzyme encoded by the gene cloned in the bacterial plasmid, usually the following steps
are performed: growth of the cells in an appropriate media until a certain cell
concentration is attained, induction of the expression of the cloned gene by a specific
inducer, cultivation for a certain time of the cells in the presence of the inducer, and the
purification of the synthesized enzyme by a general or specific technique. For the
expression of Taq DNA Polymerase I cloned in E. coli BL21 strain 3 factors were
considered, each with 2 levels (see Table ). The factor for the 23 factorial design (that
conducts to 8 experiments) are: A - the cell concentration before initiation of the
expression, considered as the optical density at 620 nm of the culture media; B - the
induction time after addition of the inducer to the culture media, and C - the temperature
of the lysis of the cells.

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 FACTORIAL DESIGN APPLIED IN MOLECULAR BIOLOGY EXPERIMENTS

Table II. The matrix design and the decoded factors for a DOE 23
factorial design
Runs Matrix design Decoded factors
A B C OD620nm Time.Ind Temp.Lys
1 - - - 0.4 12 75
2 - - + 0.4 16 85
3 + - + 0.8 16 85
4 + + + 0.8 16 85
5 - + + 0.4 16 85
6 - + - 0.4 12 75
7 + - - 0.8 12 75
8 + + - 0.8 12 75

To determine the magnitude and the importance of an effect upon the response
variable (in our case the concentration of protein in the final protein extract) a Pareto
chart was constructed. Pareto chart of the effects is used to determine the amplitude and
the significance of an effect. The chart displays the absolute value of the effects and
draws a reference line on the chart. As it can be seen from Figure 4 (left panel) the most
important effect is done by the temperature of the cell lysis step. The length of the period
of culture in the presence of the inducer (IPTG) also influences the concentration of the
expressed enzyme. At a lesser extent, a combination between the concentrations of the
cells before the addition of the inducer with the period of culture with IPTG also
influences the concentration of the enzyme in the final extract. With other words the
factors C, B and A*B are significant for the response variable.
The same conclusion can be obtained when the analysis of variance, at α < 0.05,
is applied to the series of experiments. Only for the factors C - Temp.Lys, B – Time.Ind
and A*B - DO620nm*Time.Ind, the values for p statistic were smaller than 0.05,
indicating the fact that these factors are significant.
Source DF Adj SS Adj MS F-Value P-Value
Model 8 14144247 1768031 8.65 0.005
Blocks 1 1278 1278 0.01 0.939
Linear 3 10998188 3666063 17.94 0.001
DO620nm 1 790766 790766 3.87 0.090
Time.Ind 1 4860923 4860923 23.78 0.002
Temp.Lys 1 5346500 5346500 26.16 0.001
2-Way Interactions 3 2638193 879398 4.30 0.051
DO620nm*Time.Ind 1 2024218 2024218 9.90 0.016
DO620nm*Temp.Lys 1 1278 1278 0.01 0.939
Time.Ind*Temp.Lys 1 612698 612698 3.00 0.127
3-Way Interactions 1 506588 506588 2.48 0.159
DO620nm*Time.Ind*Temp.Lys 1 506588 506588 2.48 0.159

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 M E N G H I U G . , C U T I TA R M . , E T A L .

Figure 4. The Pareto chart of the 23 factorial design used for the expression of Taq DNA polymerase
(left panel) and the normal probability plot of the effects (right panel)
To show the orientation of the effect a normal probability plot of the effect was
presented in the right panel of Figure 4. In this kind of plots the most important effects
are presented at the larger distance from the reference line. If the effect is in direct
correlation with the variation of the response, it is on the right side of the reference line.
From this plot one can see that increasing the temperature of the cell lysis step the
concentration of the expressed enzyme also increase, while when the period of the
culture in the presence of the inducer is augmented the enzyme concentration will
decrease. With other words, the effect of these two factors is opposite.
To highlight the interactions between the factors an interaction plot was
realized. As Figure 5 shows, there are some interactions between the considered factors.
For example, there is an interaction between the time of growing of the cells in the
presence of the inducer and the temperature of the cell lysis step. Parallel lines in an
interaction plot indicate no interaction, as is the case between the period of the
cultivation in the absence of inducer and the temperature of the cell lysis. An interaction
can be evidenced by the fact that the lines are not parallel. The greater the difference in
slope between the lines, the higher the degree of interaction. There is a stronger
interaction between DO620*Tim.Ind as compared with interaction Time.Ind * Temp.Lys.

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 FACTORIAL DESIGN APPLIED IN MOLECULAR BIOLOGY EXPERIMENTS

Figure 5. The interaction plots for 23 factorial design having as response variable the concentration of
the expressed enzyme in the final extract
Considering that the optimal condition can be found somewhere between the
lowest and highest values of the factors, one can use contour plots to visualize 2D-plots,
that are easy to understand than 3D plots. The 2D-contour plots show how dependent
variable relates with 2 independent variables, when the other independent variables are
fixed at a certain value. For example, considering as optimal the concentration of the
protein in the range 7000 – 8000 µg/mL, one can obtain the contour plot of variation of
Tim.Ind versus DO620nm and also the variation of Temp.Lys versus DO620nm (Figure
6). These plots show how can be modified the two factors to stay in the considered range
of the response variable. That means, one may obtain the same concentration of the
enzyme when the optical density at 620 nm range between 0.43 – 0.7 and the time of
induction between 12.5 – 13.5, but increasing one factor the other should be decreased.
Considering the other pair of factors, DO620nm and Temp.Lys, when the value of one is
increased, the level of the other should also be increased if the same concentration of the
enzyme has to be obtained.

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 M E N G H I U G . , C U T I TA R M . , E T A L .

Figure 6. The contour plots showing in a 2-D representation the correlation between the values of
two factors (independent variables) when other factors are fixed at a desired value.

Figure 7. Agarose-gel
electrophoresis of GOx gene
included in pCTcon2. Lines 1 and 3 –
for PCR was used commercial Taq
Polymerase (positive control). Line 2
negative control. Lines 4-7 samples
of the expressed Taq DNA
Polymerase

In Figure 7 the agarose gel electrophoresis of PCR products obtained by using

the purified polymerase (for lines 4 to 7) are presented. In the lines 1 and 3 the PCR was
performed with commercial Taq polymerase. This electrophoresis proves that the enzyme
produced by the expression of the gene included in the plasmid pET22 is Taq DNA
polymerase and the enzyme has an activity comparable with the commercial product.

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 FACTORIAL DESIGN APPLIED IN MOLECULAR BIOLOGY EXPERIMENTS

CONCLUSION

Two examples of the use of factorial design in molecular biology laboratory are
presented, one with 2 factors each with 3 levels and the other with 3 factors each with 2
levels. As applications, these factorial design plans were used to a protocol to purify the
plasmid pET22 containing the gene Taq DNA polymerase I, in a 32 factorial design and
in a protocol of expression of this gene in order to produce active enzyme, in a 23
factorial design. The factors that significantly influence the response were evidenced and
the interactions between the factors were emphasized.

ACKNOWLEDGEMENT

The authors thanks to Dr. R. Ostafe from RWTH Aachen University and to Dr.
R. Prodanovic, from Belgrade University for valuable help in designing and training of
the experimental part of the work. G. Menghiu acknowledge that this work was
supported by the strategic grant POSDRU/159/1.5/S/137750, Project “Doctoral and
Postdoctoral programs support for increased competitiveness in Exact Sciences research”
cofinanced by the European Social Found within the Sectorial Operational Program
Human Resources Development 2007 – 2013.

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