Molecular basis of agrobacterium mediated gene transfer into mammalian cells 2

Chapter 2. Materials and Methods
2.1. Bacterial strains, plasmids, primers, media and antibiotics
Bacterial strains and plasmids used in this study are listed in Table 2.1.
Preparations of media used in this study for the growth of bacterial strains were
formulated as listed in Table 2.2. For long-term storage, the bacteria were kept in LB
with 50% glycerol at –80°C.
Escherichia coli strains were grown at 37°C in LB (Sambrook et al., 1989) and
Agrobacterium tumefaciens strains were grown at 28°C in MG/L, AB or IB media
(Cangelosi et al., 1991) supplemented with the appropriate antibiotics when
necessary. Rhizobium meliloti (Finan et al., 1986) cells were grown in LB/MC
medium supplemented with 10 µg/ml tetracycline when necessary (Glazebrook and
Walker, 1991). Plasmid DNA was introduced into A. tumefaciens strains or
Rhizobium meliloti by electroporation (Ditta, 1980; Glazebrook, 1991). The
preparation and concentration of antibiotics and other solutions used in this study are
listed in Table 2.3.
2.2. Cell culture and culture medium
Mammalian and plant cell lines used in this study are listed in Table 2.4.
2.2.1. Mammalian cell culture
2.2.1.1. Dulbecco’s Modified Eagle’s Medium (DMEM)
DMEM was used to culture mammalian cells such as human adenocarcinoma
cell line (HeLa), human embryonic kidney (HEK) EcoPack2-293 cells and PT67 mice

46
Table 2.1. Bacterial strains and plasmids
Bacterial strain
or plasmid
Relevant characteristic(s)

Source or reference

Strains

Escherichia coli

DH5α
EndA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1
∆(argF-lacZYA)U169 φ80dlacZ ∆Μ15
Bethesda Research
Laboratories
MT607
Pro-82 thi-1 hsdR17 supE44 end44 endA1
recA56
Finan et al, 1986
MT616 MT607(pKR600), mobilizer Finan et al, 1986
S17.1(λpir) λpir lysogen, recA, thi, pro, hsdR
-
M
+
, RP4:2-
Tc:Mu:Km
R
, Tn7, Tp
R
, Sm
R

De Lorenzo et al.,
1993

Agrobacterium
tumefaciens

C58 Wild type, nopaline-type pTiC58 plasmid Laboratory
collection
A348 A136 (pTiA6NC) (octopine-type) Garfinkel et al.,
1981
A136 C58 cured of pTiC58, Rf
R
, NaI
R
Watson et al, 1975
Mx243 A136 containing pTiA6 virB
1
::Tn3-HoHo1;
virB
1
-

Stachel and Nester,
1986
Mx226 A136 containing pTiA6 virA::Tn3-HoHo1; virA
-
Stachel and Nester,
1986
Mx306 A136 containing pTiA6 virD
1
::Tn3-HoHo1; virD
-
Stachel and Nester,

1986
Mx358 A136 containing pTiA6 virE
1
::Tn3-HoHo1; virE
-
Stachel and Nester,
1986
Mx363 A136 containing pTiA6 virG
1
::Tn3-HoHo1; virG
-
Stachel and Nester,
1986
CGI1 Derivative of C58 in which aopB was disrupted
by the GFP-tagged mini-Tn5 transposon

This study
A6340 A6007 chvG
340
::TnphoA; chvG
-
Cangelosi et al.,
1991
A6880 A6007 chvH
340
::TnphoA; chvH
-
Cangelosi et al.,
1991
AG6 A348 catalase::mini-Tn5; katA

-
Xu and Pan, 2000

47
GMI9023 C58 cured of pTiC58 and pAtC58

Truchet et al., 1984
A136::Tn5 A136 derivative with mini-Tn5 insertion This study
A348::Tn5 A348 derivative with mini-Tn5 insertion This study
GMI::Tn5 GMI9023 derivative with mini-Tn5 insertion This study
Rhizobium
meliloti

RCR2011 Same as SU47 wild type Charles and Nester,
1993

Plasmids


pTZ19R Cloning vector, ColE1 oriV bla, Amp
R
US Biochemical
pLEGFP-C1 Expression vector, ColE1 oriV, Amp
R
Clontech
pSW172 Broad-host-range IncP plasmid containing P
lac

and downstream polylinker sequence, Tc
R

Chen and Winans,
1991
pBSL202 Plasmid harboring mini-Tn5 transposon, Gm
R
,
Amp
R
Suarez et al., 1997

pCB301 Broad-host-range plasmid derived from pBIN19,
Km
R

Xiang et al., 1999
pQM45 pCB301 carrying a 3 kb neoegfp fragment from
pNEOEGFP between T-borders
This study
pQM49 pSW172 carrying a 0.8 kb gfp fragment from
pAG408, Tc
R
This study
pQM52 pCB301 harboring pLEGFPC-1 between T-
borders, Amp
R
This study
pQM54 pSW172 digested with StuI and ligated to
pLEGFPC-1digested with XmnI, Tc
R
This study
pQM61 pBSL202 harboring pLEGFPC-1 between its

insertion sequences, Amp
R

This study


48
Table 2.2. Media preparation
Media or solutions Preparation
a, b
Reference
LB (Luria broth) Tryptone, 10 g; yeast extract, 5 g; NaCl, 10
g; pH 7.5
Sambrook et al.,
1989
SOB Tryptone, 20 g; yeast extract, 5 g; NaCl, 0.5
g; 10 ml of 250 mM KCl; pH 7.0,sterilize
by autoclaving and add 5ml of filter-
sterilized 2 M MgCl
2
.
Sambrook et al.,
1989
TB 10 mM PIPS, 55 mM MnCl
2
, 15 mM
CaCl
2
, 250mM KCl;

MG/L LB, 500 ml; mannitol, 10 g; g sodium
glutamate, 2.32; KH
2
PO
4
, 0.5 g; NaCl, 0.2
g; MgSO
4
.
7H
2
O, 0.2 g; biotin, 2 µg; pH
7.0.
Cangelosi et al.,
1991
AB (Minimal
medium)
20 × AB salts, 50 ml; 20 × AB buffer, 50
ml; 0.5% glucose 900 ml (autoclaved
separately before mix together).
Cangelosi et al.,
1991
IB (Induction
Medium)
20 × AB salts, 50 ml; 20 × AB buffer, 1 ml;
0.5 M MES (pH 5.5), 8 ml; 30% glucose,
60 ml (autoclaved separately before mix
together).
Cangelosi et al.,
1991

20 × AB salts
NH
4
Cl, 20 g; MgSO
4
.
7H
2
O, 6 g; KCl, 3 g;
CaCl
2
, 0.2 g; Fe SO
4
.
7H
2
O, 50 mg.
Cangelosi et al.,
1991
20 × AB buffer
K
2
HPO
4
, 60 g; NaH
2
PO
4
,

23 g; pH7.0. Cangelosi et al.,

49
1991
0.5 M MES MES, 97.6 g; pH5.5. Cangelosi et al.,
1991
1000 × AS
c
14.6 mg/ml AS in DMSO. Sambrook et al.,
1989
MS medium

Murashige and Skoog salts and vitamin
mixture, 4.42g; sucrose, 30g; 2, 4-D (0.1
mg/ml), 2ml
Murashige and
Skoog, 1962
a
Preparation for 1 liter, and sterilized by autoclaving;
b
For solid media, 1.5% agar
was added;
c
no autoclaving is necessary.

50
Table 2.3. Antibiotics and other stock solutions used in this study

Name Preparations Stock
Concentration

(Con.) (mg/ml)
Working Con.
in E. coli
(µg/ml)
Working Con.
in A. umefaciens
(µg/ml)


ampicillin
(Amp)
Dissolved in
dH
2
O, filter
sterilized
100 100
kanamycin
(Km)
Same as above 100 50 100
Carbenicillin
(Cb)
Same as above 100 100 100
gentamycin
(Gm)
Same as above 50 10 50
tetracycline (Tc) Dissolved in
absolute ethanol
5 10 5
chloramphenical

(Chl)
Same as above 34 17

Acetosyringone Dissolved in
dimethyl
sulfoxide.
100 mM
100 µm

IPTG Dissolved in
dH
2
O, filter
sterilized
24 24 24

X-Gal Dissolved in
dimethyl
sulfoxide
20 20 20
Proteinase K Dissolved in
dH
2
O
20 50 50

RNase Dissolved in
dH
2
O

10 20 20



51
Table 2.4. Mammalian and plant cell lines
cell line
Relevant characteristic(s)

Source or reference
BY-2 Nicotiana tabacum L. cv. Bright Yellow
2 callus suspension cells
Laboratory collection
HeLa human epithelial cells from a fatal
cervical carcinoma transformed by
human papillomavirus 18
Laboratory collection
EcoPack2-293 An ecotropic, HEK 293-based
packaging cell line ideal for transiently
or stably producing virus capable of
infecting mouse and rat cells.
Clontech

RETROPACKTM
PT67
A dualtropic, NIH 3T3-based packaging
cell line ideal for stably producing virus
capable of infecting a broad range of
mammalian cell types.
Clontech

52
cells (Clontech). It contains 25 mM HEPES, 4mM L-glutamine, 4.5g/l glucose, 10%
(v/v) heat-inactivated fetal bovine serum, 100 units/ml penicillin and 100µg/ml
streptomycin. For the culture of HEK-293 and PT67 cells, DMEM was supplemented
with 1nM sodium pyruvate for optimal growth. All tissue culture reagents used were
obtained from Sigma.
2.2.1.2. Mammalian cell culture and subculture
HeLa, EcoPack2-293 and PT67 cells were grown in DMEM at 37°C in a 5 %
(v/v) CO
2
incubator. Cells were maintained in 75 cm
2
flasks and subcultured at least
once every 5 days by trypsin/EDTA treatment and at a dilution of 1:4 in fresh
medium.
2.2.2. Plant cell culture and subculture
Tobacco BY2 callus (N. tabacum Bright Yellow 2) cells were maintained on
solid Murashige and Skoog’s medium (MS; Murashige and Skoog, 1962)
supplemented with 3% sucrose and 0.2 mg/ml 2,4-D. For using in A. tumefaciens
mediated transformation and attachment assay, the BY2 cells were grown in liquid
MS medium at room temperature with shaking at 100 rpm and were subcultured every
week with a 5 % inoculum.
2.3. DNA manipulations
2.3.1. Plasmid DNA preparation
Plasmid DNA was prepared following the method described previously with
some modifications (Sambrook et al., 1989). Briefly, E. coli cells from 2 ml of
overnight culture were collected by centrifugation at 10, 000 rpm (Eppendorf 5417C)

53
for 1 min. The cell pellet was resuspended in 100 µl of ice-cold solution I (50mM
glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) thoroughly by vigorous vortex.
Then, 200 µl of freshly prepared solution II (0.2 N NaOH, 1% SDS) was added and
the contents were mixed by inverting gently for 4-6 times. After the addition of 150
µl of Solution III (3 M potassium, 5 M acetate), the mixture was inverted for 4-6
times to disperse Solution III through the viscous bacterial lysate. The lysate was
extracted with equal volume of chloroform once by centrifuging at 14, 000 rpm
(Eppendorf 5417C) for 5 min. The supernatant was then transferred to a clean
eppendorf tube. To precipitate the plasmid DNA, 2 volumes of ethanol was added
and the mixture were centrifuged as above. The DNA pellet was washed once with
70% ethanol and dried in a vacuum concentrator. The extracted plasmid DNA was
dissolved in 20 µl of sterile water and stored at -20 °C, ready for subsequent use after
thawing.
2.3.2. Genomic DNA preparation from Agrobacterium
Genomic DNA of Agrobacterium was prepared according to Charles and Nester
(Charles and Nester, 1993). Cells from 100 ml of overnight culture were harvested by
centrifugation at 3000 rpm for 5 min. The cells were washed once with 4 ml of TES
(10 mM Tris-HCl, 25 mM EDTA, 150 mM NaCl, pH 8.0) and resuspended in 4 ml of
TE buffer (10 mM Tris-HCl, 25 mM EDTA, pH 8.0). To lyse the cells, 500 µl of 5 M
NaCl, 500 µl of proteinase K (5 mg/ml), and 500 µl of 10% SDS were add to the cell
suspension and then incubated at 68 °C for 30 min. The lysate was extracted once
with 1:1 phenol-chloroform and then chloroform alone. To precipitate genomic DNA,
7.5 M ammonium acetate was added to the final concentration of 2 M and then 2
volumes of ethanol were added. The DNA pellet was washed once with 70% ethanol

54
and vacuum dried. Genomic DNA was dissolved in 500 µl of distilled water and
stored at 4 °C.
2.3.3. DNA digestion and ligation

DNA digestion and ligation were conducted following the instructions of the
manufacturers supplying the enzymes. Digestion reaction systems comprised of
buffer, enzyme, DNA and water, and incubated at 37 °C for 1 hour to overnight as
required. For vectors digested with a single enzyme, dephosphorylation was carried
out by adding 0.5 µl of shrimp alkaline phosphatase into the digestion mixture.
Digested vectors and gene fragments used for ligation were cleaned using QIAGEN
gel extraction kit. Ligation was carried out by incubating the mixture of T4 DNA
ligase, vector DNA, insertion DNA, ligase buffer and water at room temperature for 4
h or overnight.
2.3.4. Polymerase chain reaction (PCR)
Polymerase chain reaction was carried out using a thermocycle (Applied
Biosystem) in a thin wall PCR tube with a volume of 200 µl. The reaction mixture
usually contained the following components in a final volume of 50 µl:

55

10 × PCR buffer (without MgCl
2
) 5 µl
25 mM MgCl
2

3 µl
Primer 1 (10 pmol/µl) 2 µl
Primer 2 (10 pmol/µl) 2 µl
dNTPs (10 mM each)
1 µl
Template DNA 20-100 ng
Taq DNA polymerase
1 µl (1 unit)

Add distilled water to a final volume of 50 µl


The PCR was run using the following program:
1 cycle
95 °C for 1 min
30 cycles
95 °C for 30 seconds

Annealing at (Tm-5) °C for 30 seconds

Extension at 72 °C for 1 min per kb
1 cycle
72 °C for 10 min


56
2.3.5. DNA gel electrophoresis and purification
DNA fragments were electrophoresized in an 1 × TAE (0.04 M Tris-acetate,
0.001 M EDTA, pH 8.0) agarose gel along with a standard DNA marker (Fermentas).
Digested DNA vectors and fragments from genomic DNA or PCR products to be used
for ligation and transformation reaction were usually purified with QIAquick Gel
Extraction Kit (QIAGEN) following the instructions provided by the manufacturer.
Briefly, DNA was separated in an 1% agarose gel. The gel slice containing the
desired DNA bands were excised and transferred to a pre-weighted eppendorf tube.
Then 3 gel volumes (100 mg gel ≈ 100 µl) of buffer QG were added and the tube was
incubated in an 55 °C waterbath for 5-10 min to dissolve the gel completely. For
DNA fragments larger than 4 kb or smaller than 500 bp, 1 gel volume of isopropanol
was added. The mixture was transferred to a QIAquick spin column in an 2-ml
collection tube. The binding of DNA to the column was achieved by centrifugation

for 1 min at 14, 000 rpm (Eppendorf 5417C). The column was then washed once with
750 µl of buffer PE with one additional centrifugation to remove residual ethanol.
The column was placed into a clean 1.5-ml centrifuge tube. To elute DNA, 50 µl of
sterile water was applied to the center of the column membrane and the column was
centrifuged at 14, 000 rpm (Eppendorf 5417C) for 1 min.
2.3.6. Preparation of competent cells
E. coli DH5α was routinely used as the host for cloning experiments unless
otherwise specified. High efficient competent cells were prepared as described
previously (Inoue et al, 1990). E. coli cells were streaked from frozen stock and
cultured overnight on an LB plate at 37 °C. Then several colonies were picked and
inoculated into 100 ml of SOB medium in a 1-liter conical flask. The cells were

57
cultured at room temperature (about 19°C) with vigorous shaking (250 rpm) to an
OD
600
of 0.5-0.7. The cells were chilled on ice for 10 min before they were collected
by centrifugation at 2600 rpm (Eppendorf 5810R) for 5 min at 4 °C. The cell pellets
were resuspended in 30 ml of ice-cold TB buffer (10 mM PIPES, 55 mM MnCl
2
, 15
mM CaCl
2
, 250mM KCl, pH 6.7; all components except MnCl
2
were dissolved and
autoclaved; 1M MnCl
2
solution was filter-sterilized and added to make TB buffer;
store at 4 °C) and then incubated on ice for 10 min. Cells were collected by

centrifugation as above and resuspended in 5 ml of ice-cold TB buffer. Thereafter,
DMSO was added to a final concentration of 7% and the cell suspension was
aliquoted into pre-cooled sterile eppendorf tubes at 100 µl each. The competent cells
were kept at -80 °C until needed.
2.3.7. Transformation of E. coli
A plasmid or a ligation reaction product was introduced into E. coli by
transformation for amplification or screening (Sambrook et al, 1989). A frozen
competent cell (100 µl) was thawed on ice. Plasmid (50-100 ng in 10µl or less) or
ligation product (10 µl) was added and the contents of the tube were mixed by gently
tapping the tube a few times. The tube was then incubated on ice for 30 minutes. The
mixture of cells and DNA were heat-shocked at 42 °C for 90 seconds. After chilling
the cells on ice for 2 min, 900 µl of fresh LB medium were added. The cultures were
incubated at 37 °C for 30 min with agitation. The cells were collected and spread
onto a LB agar plate containing appropriate antibiotic(s) or substrate(s). Colonies
usually appear after 12-16 hr of incubation at 37 °C.

58
2.3.8. Shuttling of broad-range plasmids between E. coli and A. tumefaciens or R.
meliloti
Introduction of broad-range plasmids into Agrobacterium tumefaciens or
Rhizobium meliloti were carried out by triparental mating (Ditta et al., 1980) or
electroporation (Cangelosi et al., 1991). Triparental mating was conducted by mixing
equal proportions of helper strain MT616, donor strain and recipient strain cells
together on MG/L (for Agrobacterium tumefaciens) or LB/MC (for Rhizobium
meliloti) agar plate and incubating the plate overnight at 28°C. A small amount of
the mating mixture was picked and streaked onto an AB or M9/sucrose agar plate
containing appropriate antibiotics. The A. tumefaciens exconjugates usually appeared
after 2-3 days of incubation while E. coli could not grow on AB or M9/sucrose plates.
For triparental mating involving the transfer of plasimd from Agrobacterium into E.
coli, a similar procedure was performed. The mating mixture of three parent strains

was incubated on an LB plate overnight and then streaked onto an LB plate containing
the appropriate antibiotics to select for E.coli containing the plasmid. The plate was
cultured overnight at 37 °C since neither A. tumefaciens nor Rhizobium could grow at
37 °C.
Electroporation was also used in this study to introduce a plasmid into
Agrobacterium. Electrocompetent Agrobacterium cells were prepared as follows.
Cells cultured overnight at 28 °C were scraped from the plate with a sterile wooden
stick and then transferred into a sterile eppendorf tube. The cells were washed once
with ice-cold water and once with ice-cold 15% glycerol. The cell pellet was
resuspended in 50-100 µl of ice-cold 15% glycerol and then plasmid DNA (50-100 ng
in 10 µl or less of water) was added. The mixture of cells and DNA was transferred

59
to a chilled BioRad electroporation cuvette and kept on ice for 10 min. Gene Pulser II
Electroporation System (BioRad) was set to the 25-µF capacitor, voltage of 2.5 kV
and controller unit of 400 ohms. The outside of the cuvette was wiped with tissue
paper to get rid of moisture before the cuvette was slide into the shocking chamber
base. The cells were usually pulsed for 8-10 milli-seconds. Then, 1 ml of MG/L
medium was added and the mixture was transferred to an 15-ml culture tube. After
culturing at 28 °C for 1h, the cells were collected and spread onto an MG/L plate
containing the selectable antibiotics. Colonies usually appeared three days later.
2.4. RNA manipulations
2.4.1. RNA isolation from mammalian cells
Total RNA of mammalian cells was prepared using TRIZOL Reagent
(GIBCO/Life Technologies,

Grand Island, NY) according to the manufacturer’s
instructions. In brief, mammalian cells from one 75 cm
2
flask were washed once with

10 ml of PBS before 2 ml of TRIZOL Reagent were added. The homogenized sample
was then vortexed for 30 s and incubated at room temperature for 5 min. Residual
protein was removed after the steps of addition of 400 µl of chloroform, mixing for 30
s, incubation at room temperature for 3 min, and centrifugation for 15 min at 12000 ×
g and 4 °C. The RNA in the colorless aqueous phase was precipitated in 1 ml of
isopropanol by mixing for 15 s, incubation for 10 min at room temperature, and
centrifugation for 10 min at 12000×g and 4 °C. The resulting RNA pellet was washed
with 1 ml of 75% ethanol and centrifuged for 5 min at 7500 × g and 4 °C. The RNA
pellet was air dried, resuspended in DEPC-treated water, and stored at -80 °C. The
extracted RNA was treated with DNase before the RT-PCR was conducted.

60
2.4.2. RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) was carried out
using QIAGEN onestep RT-PCR kit (QIAGEN). The 25 µl RT-PCR reaction mixture
was comprised of the following components:
5 × RT-PCR buffer 5 µl
RNase-free water
11 µl
Primer 1 (10 pmol/µl) 0.5 µl
Primer 2 (10 pmol/µl) 0.5 µl
dNTP Mix
1 µl
RNase inhibitor
1 µl
RNA template
5 µl
QIAGEN Onestep RT-PCR Enzyme Mix
1 µl

61
The RT-PCR were run using the following program:
1 cycle
50 °C for 30 min
1 cycle
95 °C for 15 min
40 cycles
94 °C for 1 min

Annealing at (Tm-5) °C for 30 seconds

Extension at 72 °C for 1 min per kb
1 cycle
72 °C for 10 min

2.5. Southern blot analysis
Southern blot analysis was carried out using the ECL random prime labeling and
detection system (Amersham life science) following the instructions of the
manufacturer.
2.5.1. Labeling of probes with fluorescein
Probes were labeled randomly with fluorescein (Amersham) as described below.
Plasmid DNA was digested with the appropriate restriction enzyme(s). The target
DNA fragment was isolated from the agarose gel after electrophesis and purified by
QIAquick Gel Extraction Kit (QIAGEN). The DNA fragment (50-500 ng in 34 µl of
water) was denatured by boiling for 5 min and chilled on ice. Then the nucleotide
mix (10µl), random primers (5 µl) and Klenow enzyme solution (1 µl) were added
and mixed by gently pipetting. The reaction mix was incubated at 37 °C for one hour

62

and terminated by the addition of EDTA to a final concentration of 20 mM. The
probes could be stored at – 30 °C in the dark for one year till needed.
2.5.2. Membrane blot preparation
Genomic DNA of A. tumefaciens or Rhizobium meliloti was digested and then
separated in an 1.0% agarose gel. The agarose gel was depurinated by incubation in
the depurination solution (0.25 M HCl) at room temperature for 10 min with agitation
and followed by denaturation (denaturation solution: 1.5 M NaCl, 0.5 M NaOH) for
25 min and neutralization (neutralization solution: 1.5 M NaCl, 0.5M Tris-HCl, pH
7.5) for 30 min. The PosiBlot 30-30 Pressure Blotter (Stratagene) was assembled
with the sponge (pre-wet by 10 × SSC), filter paper, mask with window, agarose gel
and nylon membrane inside from up to bottom. The DNA was transferred onto the
nylon membrane at a constant pressure of 60-75 Hg for 1-2 h by using 10 × SSC (0.15
M Na
3
Citrate, 1.5 M NaCl) as the transfer buffer. Thereafter, the DNA fragments
were fixed on the membrane by UV crosslinking for 2 min.
2.5.3. Hybridization and stringent wash
The blots were put into 15 ml of hybridization buffer (5 × SSC, 0.1% SDS, 5%
dextran sulphate, 20 × dilution of liquid block) preheated to 65 °C and prehybridized
for 1 h with rolling in a hybridization oven (HYBAID). Then, denatured probe were
added and hybridization was conducted at 65 °C for overnight as above.
The membrane was transferred into an excess volume of washing solution
(1×SSC, 0.1% SDS) and washed for 15 min twice at 65 °C with gentle agitation. One
further wash was carried out using 0.5 × SSC, 0.1% SDS as the washing solution.

63
2.5.4. Blocking, antibody incubation and washing
The above treated blot was then transferred to a clean container and rinsed with
buffer A (100 mM Tris-HCl, 600 mM NaCl, pH 7.5) for 1 min. Blocking was
conducted by incubating the membrane in an 20-fold diluted of liquid block in buffer

A (100 mM Tris-HCl, 600 mM NaCl, pH 7.5) for 30 min at room temperature. The
blot was then put in an appropriately sized container and incubated in diluted
conjugate solution (1000-fold diluted anti-fluorescein-HRP conjugate in freshly
prepared 0.5% BSA in buffer A) for 30 min. The unbound conjugate was removed by
three washes in excess of 0.1% Tween-20 in buffer A for 10 min each.
2.5.5. Signal generation and detection
The washed blot was placed in premixed detection solution I and II at equal
volume and incubated for 1 min. The blot was then transferred to a film cassette and
a sheet of autoradiograpy film was exposed for an appropriate length of time before
the film was developed in a KODAK developing machine.
2.6. Protein techniques
2.6.1. Buffers for protein manipulations
Buffers used in protein manipulations are listed in Table 2.5.
2.6.2. SDS-PAGE gel electrophoresis
Protein profiles were analyzed using SDS-PAGE (Laemmli, 1970) based on
molecular weight. The electrophoresis apparatus used was the Mini-Protean III
Electrophoresis Cell (BioRad). The apparatus was assembled according to the
instructions provided by the manufacturer. The monomer stock solution of

64
Table 2.5. Buffers used in protein manipulations
Name Components (for 1 L) pH adjustment
0.2 M Tris base
1.37 M Sodium chloride
10 × Tris-buffered saline
(10 × TBS)
38 ml 1M Hydrochloric acid
Adjust pH to 7.6
1 × TBST 0.1% Tween-20 (v/v) in 1 × TBS

0.25 M Tris
1.92 M Glycine
10 × Tank buffer
0.1% SDS
No need to check
pH
48 mM Tris
38 mM Glycine
0.37 g SDS
10 × Transfer buffer

20% Methanol
Adjust pH to 8.3
4 × Separating gel buffer
1.5 M Tris-HCl
Adjust pH to
8.8
4 × Stacking gel buffer
0.5 M Tris-HCl
Adjust pH to
6.8
50 mM Tris-HCl (pH 6.8)
100 mM Dithiothreitol
2% SDS
0.1% Bromophenol blue
1 × SDS gel-loading buffer
20% Glycerol

0.25 g Coomassie Brilliant blue R
(Gibco)

400 ml Methanol
Staining solution
70 ml Acetic acid

400 ml Methanol Destaining solution I
70 ml Acetic acid

70 ml Acetic acid Destaining solution II
50 ml Methanol

65
acrylamide/bis-acrylamide (30.8%T/2.7%C) was prepared as described in Molecular
Cloning (Sambrook et al., 1989) and stored in dark at 4 °C. Ammonium Persulfate
(APS) (10%) solution was freshly prepared before each use. Separating gel buffer (4
×, 1.5 M Tris-HCl, pH 8.8) and stacking gel buffer (4 ×, 0.5 M Tris-HCl, pH 6.8)
were stored at room temperature. Tank buffer was prepared as a 10 × stock solution
(0.25 M Tris-HCl, 1.92 M glycine, 1% SDS, pH8.3) and stored at room temperature.
Gel loading buffer (2 ×, 100 mM Tris-HCl, pH6.8, 4% SDS, 0.2% bromophenol blue
and 20% glycerol, 0.2 M DTT) was prepared without DTT and stored at room
temperature. DTT was added from a 2 M stock solution and stored at -20°C before
use. The preparation of polyacrylamide gel and the separation of proteins were
performed following the instructions of Hoefer Scientific Instruments (Protein
electrophoresis-applications guide, 1994). In this study, 12% PAGE gel was used for
the analysis of proteins unless otherwise specified.
2.6.3. Staining of SDS-PAGE separated proteins with standard Coomassie blue
SDS-PAGE separated proteins were stained according to the instructions of
Hoefer Scientific Instruments (Protein electrophoresis-applications guide, 1994). The
gel was placed in the staining solution and shaked at low speed for 1 h. The staining
solution was then discarded and replaced with destaining solution I. After the gel had
been destained for 30 min, destaining solution I was removed and replaced with

destaining solution II. The destaining solution II was changed twice a day until the
gel background was clear.
2.6.4. Western blot analysis
The sample was mixed with equal volume of 2 × loading dye buffer (Laemmli,
1970), and boiled in a water bath for 5-10 min. After cooling down, the sample was

66
loaded into a SDS/12% polyacrylamide gel, and separated at a constant voltage of
100V. The protein was transferred to an Immun-Blot
TM
PVDF membranes (Bio-Rad)
from the gel in mini gel transfer system for 4 hr to overnight at 200 mA before the
non-specific binding sites were blocked by immersing the membrane in 10 % non-fat
milk (Nestle) in TBST for 2 h at room temperature on an orbital shaker. The
membrane was then washed in TBST buffer for 3×10 min, and incubated in the
diluted primary antibody for 1 h at room temperature. The membrane was washed
three times as above before incubation in the diluted secondary antibody for 1 h at
room temperature. After washing thoroughly as above, the membrane was processed
for signal detection according to the recommendations of the manufacturer
(Amersham).
2.7. Transposon tagging
A. tumefaciens A348 and E. coli S17.1(
λ
pir) with target transposon were freshly
grown as the recipient and donor respectively. The cell density was 5 × 10
7
cells per
ml for both the donor and recipient, but the ratio of the cell suspension volume for the
donor to the recipient was 1:4. After the mixture was spotted on the LB plates, the
plates were dried for 1 h at 37°C and then incubated at 28°C for 24 h. Exconjugants

were selected on minimal medium AB plates containing 100 µg ml
-1
of gentamycin.
The plates were incubated at 28°C for 2 days to select for the transposon mutants,
since gentamycin resistance is encoded by the transposon that can replicate in E. coli
S17.1(λpir) but not in A. tumefaciens that is sensitive to gentamycin.

67
2.8. General protocol for gene transfer into mammalian cells
2.8.1. Preparation of bacteria
E. coli, A. tumefaciens and R. meliloti cells were grown overnight in LB ,MG/L
or LBMC liquid medium supplemented with the appropriate antibiotics before they
were harvested from the liquid culture by centrifugation at 10,000 g. The bacterial
cells were washed with PBS and diluted to an OD
600
of 0.2-0.3 for induction or gene
transfer assay.
For A. tumefaciens vir genes induction, cells were resuspended to an OD
600
of
0.2 to 0.3 in IB (Cangelosi et al., 1991) liquid medium in the presence of 100 µM
acetosyringone (AS). The cell cultures were incubated at 28 °C for 18-20 h with
shaking.
2.8.2. Preparation of mammalian cells
Mammalian cells were trypsinized and resupended in fresh DMEM with
antibiotics and then seeded at 1 ×10
6
cells in each well of an 6-well tissue culture
plate. The cells were then incubated at 37°C in an 5% CO
2

incubator for 18 h. About
4 h before incubation, the cells were washed once with PBS and incubated in fresh

medium without

antibiotics. For the study of the staining pattern of rhodamine
phalloidin, mammalian cells were seeded on the glass cover-slip in 6-well tissue
culture plates.
2.8.3. Agrobacterium-mediated gene transfer into mammalian cell
Mammalian cells were cocultured with A. tumefaciens by adding 200 µl of
bacterium cells (about 4x10
7
bacteria) to each of the 6 well tissue-culture plates

68
containing the mammalian cells and incubated at room temperature for 4 h. Then the
plates were transferred to an 37°C incubator with 5% CO
2
. After an overnight
incubation, the mammalian cells were washed with PBS for three times to remove the
no-specifically bound bacterial cells before they were incubated in 3 ml of complete
medium supplemented with 1 mM cefotaxime, which would kill bacterial cells.
These cells were used subsequently for later assays.
2.8.4. Attachment and internalization assays
To measure the number of bacteria adhering to the mammalian cells, the 6-well
plates were washed 3 times with PBS solution before the mammalian cells were lysed
with 1% (v/v) Triton X-100 in PBS. The number of bacteria was quantified by agar
plate culturing and counting. To measure the internalization of bacteria, the
mammalian cells were washed three times with PBS and then incubated for an
additional 2 h in tissue culture medium containing 100µg/ml gentamycin (Sigma) to

kill extracellular bacteria. After incubation, the mammalian cells were washed twice
with PBS solution and lysed with 1% Triton X-100 in PBS before the numbers of
bacteria were quantified as above. The adhesion and invasion rates were calculated
from the average of three independent experiments.
2.8.5. Observation of gene transfer events
2.8.5.1. Fluorescence microscope analysis
To detect the transient expression of egfp, the mammalian cells were examined
with a Zeiss Axioskop microscope equipped with a filter set for GFP (exciter filter BP
480nm, banner filter 510-520nm) using 40 X plan neuflor objectives 36 h after
incubation. Photographs were recorded on Fuji 100 colour film.

69
2.8.5.2. Flow cytometry analysis
EcoPack2-293 Packaging Cells were resuspended in PBS and filtered through
10 µm of nylon before examined under a FACS cytometer, using 100 mW at 488 nm.
The fluorescence intensity from 100,000 cells was recorded.
2.8.6. Confocal microscopy analysis
Mammalian cells were seeded on the glass coverslips in 6-well tissue culture
plates and incubated with the Agrobacterium harbouring pQM49, which express
gfpuv constitutively. After 4 h incubation, the mammalian cells were washed with
PBS and examined under a confocal microscope. A blue laser of excitation
wavelength

488 nm was used to view Agrobacterium cells expressing GFPuv. Optical

sectioning of 293 cells was carried out serially at 0.8 µm intervals along the apical
and basal axies of

the cells.
2.8.7. Observation of actins in mammalian cells

Rhodamine phalloidin (Molecular Probes) was used to label actin of
mammalian cells according to the manufacturer’s instructions. In brief, mammalian
cells were fixed for 10 min with PBS/3.7% paraformaldehyde at room temperature
(RT) and then washed twice with cold PBS/0.1% BSA. After permeabilization for 10
min with PBS/0.1%BSA/0.1%Triton X-100 at RT, the cells were equilibrated by
incubation with PBS/2%BSA for 10 min at RT and then incubated with phalloidin-
rhodamine (Molecular Probes, Eugene, OR) (5 µl /200 µl PBS/1%BSA) for 1 h at RT
in the dark. After staining, the coverslips were washed by PBS for 4 times. Labelled
actin was visualized under a confocal laser scanning microscope using an excitation
wavelength of 514 nm.

70