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54
CHAPTER 2
MATERIALS AND METHODS
2.1 CHEMICALS AND REAGENTS
The enzymes and reagents used in cloning experiments viz., NcoI,
XhoI, T4 DNA ligase, and Phusion™ Flash High-Fidelity PCR Master Mix,
M-MuLV reverse transcriptase, were purchased from New England Biolabs
(Manassas, USA). Taq Polymerase was procured from Fermentas (USA).
Plasmid miniprep spin kit, PCR purification kit, RNAprotect Bacteria
Reagent, RNeasy Mini Kit, RNase-Free DNase Set, random hexamers, dNTP
Mix (PCR Grade) and Proteinase K were procured from Qiagen (Germany).
Primers were procured from VBC-Biotech (Austria) and the inducer sakacin P
induction peptide (SppIP) was synthesized from GenScript (USA).
Chloramphenicol, ampicillin, kanamycin and lysozyme were bought from
Biobasic Inc (USA). Mutanolysin and 13C3- Glycerol were procured from
Sigma-Aldrich (USA). Culture media (LB and MRS) were purchased from
HiMedia Laboratories (Mumbai, India) and the individual media components
and other chemicals were obtained from either HiMedia Laboratories
(Mumbai, India) or from Merck (India). Since reuterin standard could not be
commercially procured, it was synthesized in our laboratory as described
under “Reuterin production by resting cells of L. reuteri ATCC 55730”.
2.2 CULTURE AND MAINTENANCE
The bacterial strains and plasmids used and modified in this study
are listed in Table 2.1.
55
Tab
le 2
.1 B
acte
rial
stra
ins a
nd p
lasm
id v
ecto
rs u
sed
in th
e st
udy
Stra
in o
r pl
asm
id
Des
crip
tion
Sour
ce o
r re
fere
nce
E. c
oli D
H5
Clo
ning
hos
t for
TA
ve
ctor
In
vitro
gen,
USA
E. c
oli E
C10
00
Clo
ning
hos
t for
pS
IP41
1 D
r Jan
Kok
, Uni
vers
ity
of G
roni
ngen
, N
ethe
rland
s
RB
C- T
A v
ecto
r TA
clo
ning
vec
tor
RB
C B
iosc
ienc
e C
orp.
, Ta
iwan
pSIP
411
E. c
oli-l
acto
baci
llus
shut
tle e
xpre
ssio
n ve
ctor
Sorv
ig e
t al (
2005
)
L. re
uter
i ATC
C 5
5730
H
ost
B
ioga
ia, S
wed
en
L. re
uter
i HR
2 L.
reut
eri w
ith y
qhD
Th
is st
udy
E. c
oli K
-12
MG
1655
So
urce
of y
qhD
gen
e Pr
of. T
akas
hi H
oriu
chi,
Nat
iona
l Ins
titut
e fo
r B
asic
Bio
logy
, Jap
an.
pHR
1TA
vec
tor w
ith y
qhD
Th
is st
udy
pHR
2pS
IP41
1 w
ith y
qhD
This
stud
y
L. r
eute
ri A
TCC
557
30 a
nd th
e E.
col
i stra
ins
wer
e gr
own
at 3
7 °C
in M
RS
brot
h (T
able
2.2
) an
d LB
(try
pton
e 1%
; yea
st e
xtra
ct 0
.5%
; sod
ium
chlo
ride
1%)
brot
h, r
espe
ctiv
ely.
A s
emi-d
efin
ed m
ediu
m a
s de
scrib
ed b
y
Lave
nder
et a
l., (
2001
) was
use
d fo
r met
abol
ic fl
ux a
naly
sis
with
out l
acto
se,
but w
ith g
luco
se (
27.7
5 m
M)
and
glyc
erol
(69
.38
mM
). C
asam
ino
acid
s in
the
med
ium
wer
e re
plac
ed w
ith c
asei
n en
zym
e hy
drol
ysat
e (ty
pe I
). In
addi
tion,
the
sem
idef
ined
med
ium
was
com
pose
d of
am
ino
acid
s, tra
ce
elem
ents
, vi
tam
ins,
nucl
eotid
es
and
twee
n 80
.Th
e re
com
bina
nts
wer
e
cultu
red
in m
edia
con
tain
ing
appr
opria
te a
ntib
iotic
s, am
pici
llin
(100
µg/
mL)
and
eryt
hrom
ycin
(200
µg/
mL
for E
. col
i and
5 µ
g/m
L fo
r L. r
eute
ri).
Gro
wth
was
mon
itore
d by
mea
surin
g th
e ab
sorb
ance
at
600
nm.
Cel
l dr
y w
eigh
t
56
(CD
W)
was
cal
cula
ted
from
a p
rede
term
ined
rel
atio
nshi
p be
twee
n L.
reu
teri
CD
W a
nd o
ptic
al d
ensi
ty (1
OD
600 c
orre
spon
ded
to 0
.33
g/l C
DW
).
For l
ong
term
stor
age,
E. c
oli a
nd L
. reu
teri
cel
ls w
ere
grow
n in
LB
med
ium
and
MR
S m
ediu
m re
spec
tivel
y, w
ith a
ppro
pria
te a
ntib
iotic
s at
37
ºC.
This
was
use
d to
inoc
ulat
e 10
ml o
f LB
or M
RS
and
the
cells
gro
wn
until
the
OD
600 r
each
ed 0
.8 to
1.0
(log
arith
mic
pha
se).
The
log
phas
e cu
lture
was
use
d
to m
ake
glyc
erol
(30%
) sto
cks a
nd st
ored
at -
80 ºC
.
Tab
le 2
.2 C
ompo
sitio
n of
MR
S m
ediu
m
Ingr
edie
nts
Con
cent
ratin
(g/l)
Y
east
ext
ract
5
Prot
eose
pep
tone
10
Bee
f ext
ract
10
Dip
otas
sium
pho
spha
te
2
Am
mon
ium
citr
ate
2
Sodi
um a
ceta
te
5
Mag
nesi
um su
lpha
te
0.1
Man
gane
se su
lpha
te
0.05
Poly
sorb
ate
80
1
Dex
trose
20
Fina
l pH
6.
5
57
Tab
le 2
.3 P
repa
ratio
n of
stoc
k so
lutio
ns o
f ant
ibio
tics
and
indu
cer
Rea
gent
/Che
mic
alSt
ock
Fina
l Con
cent
ratio
n
Am
pici
llin
100
mg/
ml i
n di
still
ed w
ater
10
0 µg
/ml
Eryt
hrom
ycin
10
0 m
g/m
l in
abso
lute
al
coho
l
10 m
g/m
l in
abso
lute
alc
ohol
200
µg/m
l (E.
col
i)
5µg/
ml (
L. re
uter
i)
Indu
cer (
SppI
P)
1 m
g/m
l in
dist
illed
wat
er
50 n
g/m
L
2.3
MO
LE
CU
LA
R B
IOL
OG
Y M
ET
HO
DS
2.3.
1 G
enom
ic D
NA
Isol
atio
n
The
geno
mic
DN
A o
f E.
col
i K
-12
MG
1655
stra
inw
as i
sola
ted
usin
g a
slig
htly
mod
ified
hig
h sa
lt, S
DS-
base
d m
etho
d (Z
hou
et a
l 199
6). 0
.5
ml o
f th
e ov
erni
ght c
ultu
re w
as p
elle
ted
in a
mic
roce
ntrif
uge
tube
at 1
0,00
0
rpm
for 1
0 m
in. T
he c
ell p
elle
t was
was
hed
in P
BS
buff
er (1
37 m
M N
aCl,
2.7
mM
KC
l, 2.
0 m
M K
H2P
O4,
10 m
M N
a 2H
PO4;
pH 7
.4)
and
susp
ende
d in
0.5
ml o
f TK
M I
buff
er (1
0 m
M T
ris, p
H 7
.6, 1
0 m
M K
Cl,
10 m
M M
gCl 2,
2 m
M
EDTA
). Th
e ce
lls w
ere
pelle
ted
agai
n an
d re
susp
ende
d in
TK
M II
buf
fer (
0.4
M N
aCl a
dded
to T
KM
I bu
ffer
) con
tain
ing
25
l of 1
0 m
g/m
l lys
ozym
e an
d
incu
bate
d at
37
°C f
or 1
5 m
in. T
o th
e su
spen
sion
, 50
l of
10%
SD
S w
as
adde
d, m
ixed
and
incu
bate
d at
55
°C f
or 1
0 m
in. T
hen
250
l of
5 M
NaC
l
was
add
ed t
o th
e su
spen
sion
and
mix
ed w
ell.
The
cells
wer
e pe
llete
d an
d
58
centrifugation as above. Finally the DNA was dried and resuspended in 50 l
TE buffer (10 mM Tris, 1 mM EDTA; pH 7.6).
2.3.2 Plasmid DNA Isolation
Plasmid DNA was isolated using Qiagen Mini prep kit using the
following protocol. 1.5 to 3 ml of E. coli culture was centrifuged at 8000 rpm
for 2min. Pellet was resuspended in 250 µl of buffer P1 by vortexing. 250 µl
of buffer P2 was added and mixed gently by inverting the tube 4-6 times until
the solution becomes clear. 350 µl of buffer N3 was added and mixed
immediately to form a white precipitate. The mixture was centrifuged at
13000 rpm for 10 min. Supernatant was carefully removed without any
precipitate and transferred to Qiaprep spin column. The column was
centrifuged at 13000 rpm for 60 s and the flow through was discarded. The
column was further washed with 500 µl of buffer PB and centrifuged for 60 s
at 13,000 rpm. 750 µl of buffer PE was added to the column and washed. The
column was transferred to a fresh 1.5 ml eppendorf and 50 µl elution buffer
was added, incubated for 2 to 5 min and then centrifuged to elute the plasmid
DNA.
For the isolation of plasmid from L. reuteri, Qiagen Mini prep kit
was used with the following modification: The cells in resuspension buffer,
were lysed with 30 mg/mL lysozyme (USB) and incubated at 37 ºC for
30 minutes. The rest of the procedure was done as described above.
2.3.3 RNA Isolation
RNA was isolated using RNeasy Mini Kit (Qiagen). The RNA in
the bacterium was stabilised by adding 500 µl (2 volumes) of RNA protect
bacteria reagent to 250 µl of bacterial culture. The mixture was vortexed for
5s and incubated at room temperature for 5 min. Further, it was centrifuged
59
for 10 min at 5000 x g and supernatant was decanted. The residual
supernatant was removed by gently dabbing onto paper towel. The pellet was
stored at -70 ºC for until use (upto 4 weeks). The following protocol was
adopted to isolate RNA from the processed culture.
L. reutrei cells were lysed in 100 l TE (10 mM Tris.Cl, 1mM
EDTA; pH 8.0)buffer containing 10 l proteinase K, 50 units mutanolysin
and 25 mg/ml lsyozyme and incubated at 37 ºC for 15 min. It was vortexed
for 10 s for every 2 min. 350 µl of buffer RLT was added and vortexed
vigorously. To this solution, 250 µl of absolute ethanol was added, mixed and
transferred to mini-spin column with collection tube. The sample was
centrifuged for 15 s at 8000 x g and the flow through was discarded. 350 µl of
RW1 buffer was added to the spin column and centrifuged for 15 s at 8000 x
g and flow through was discarded.
On-column DNase treatment was given, by adding DNaseI
incubation mix (10 µl of DNaseI stock solution + 140 µl of buffer RDD) to
the column and further incubating for 15 min at room temperature. 350 µl of
RW1 buffer was added to spin column and kept for 5min further centrifuged
for 15 s at 8000 x g. The collection tube was changed, and 500 µl of RPE
buffer was added to the spin column and centrifuged for 2 min at 8000 x g to
wash the spin column membrane. The mini spin column was placed in 1.5ml
tube and 50 µl of RNase free water was added to the spin column membrane.
The column was kept for 2 to 5 min and further centrifuged for 1 min at 8000
x g to elute the RNA. The isolated RNA was stored in -80 ºC until use.
2.3.4 Polymerase Chain Reaction (PCR)
PCR was performed using Taq polymerase and Phusion
polymerase. The PCR reaction mixture was made using the following
components (Table 2.4):
60
Table 2.4 components of PCR reaction
Components Final Concentration Forward Primer 500 nM
Reverse Primer 500 nM
dNTP 200 M
Template DNA 2 to 50 ng/ l
10X Polymerase buffer 1X
DNA Polymerase 1U
2.3.4.1 Cycling conditions for Taq polymerase
Initial denaturation 95 ºC 5 min
Denaturation 95 ºC 1 min
Annealing 55 - 65 ºC 1 min
Extension 72 ºC 1 min 20 sec (1 kb/min)
Final extension 72 ºC 10 min
29 cycles from step 2 to 4
2.3.4.2 Cycling conditions for Phusion polymerase
Initial denaturation 98 ºC 10 sec
Denaturation 98 ºC 3 sec
Annealing 65 ºC 5 sec
Extension 72 ºC 20 sec (1 kb/15 sec)
Final extension 72 ºC 1 min
24 cycles from step 2 to 4
61
2.3.
5 D
NA
Cle
anup
from
Aga
rose
Gel
The
band
cor
resp
ondi
ng t
o th
e de
sire
d D
NA
fra
gmen
t in
the
agar
ose
gel w
as r
apid
ly c
ut u
sing
a s
calp
el o
n a
UV
lam
p ta
ble.
The
siz
e of
the
gel s
lice
was
min
imiz
ed b
y re
mov
ing
the
extra
aga
rose
. DN
A fr
om th
e ge
l
was
pur
ified
usi
ng Q
ia q
uick
gel
ext
ract
ion
kit (
Qia
gen)
. Brie
fly th
e ag
aros
e
gel w
ith D
NA
was
sol
ubili
sed
with
buf
fer Q
G a
t 50
ºC a
nd tr
ansf
erre
d to
Qia
quic
k sp
in c
olum
n co
ntai
ning
sili
ca m
embr
ane,
whi
ch s
peci
fical
ly b
inds
to
DN
A w
hile
con
tam
inan
ts f
low
thr
ough
the
col
umn.
The
col
umn
is w
ashe
d
with
buf
fer
PE a
nd th
e pu
re D
NA
was
elu
ted
with
Tris
buf
fer
(10
mM
Tris
-
Cl,
pH8.
5) o
r wat
er.
2.3.
6 A
-tai
ling
of P
CR
Pro
duct
The
PCR
pr
oduc
t w
as
subj
ecte
d to
ge
l pu
rific
atio
n or
PC
R
purif
icat
ion
proc
ess
and
purif
ied
prod
uct w
as u
sed
for A
-taili
ng. B
riefly
in a
reac
tion
volu
me
of 1
0 µl
, 1 x
Taq
rea
ctio
n bu
ffer
, 3 µ
l (10
0 ng
) of
pur
ified
PCR
pro
duct
, 0.2
mM
dA
TP, 1
µl T
aq D
NA
pol
ymer
ase
was
add
ed a
nd th
e
mix
ture
was
incu
bate
d at
70
ºC fo
r 60
min
.
2.3.
7 PC
R P
urifi
catio
n us
ing
Spin
Col
umn
The
QIA
quic
k ge
l ext
ract
ion
kit (
Qia
gen)
bas
ed w
as u
sed
for
the
clea
nup
of D
NA
in P
CR
reac
tion
mix
ture
and
oth
er e
nzym
atic
reac
tions
. The
kit
uses
a s
pin
colu
mn
met
hod
whi
ch i
nvol
ves
the
spec
ific
bind
ing
of s
pin
colu
mn
mem
bran
e w
ith 1
00 b
p to
10
kb o
f D
NA
fra
gmen
ts, p
urify
ing
them
from
prim
ers,
poly
mer
ase,
sal
ts a
nd o
ther
com
pone
nts.
The
bind
ing
of D
NA
occu
rs a
t a p
H
7.5
and
elut
ion
is c
arrie
d ou
t hig
her p
H (p
H 8
.0).
Brie
fly, t
he
DN
A s
ampl
e is
mix
ed w
ith 5
vol
umes
of
buffe
r PB
and
tran
sfer
red
to s
pin
colu
mn
and
then
col
umn
was
was
hed
with
was
h bu
ffer
s and
the
purif
ied
DN
A
is e
lute
d us
ing
Tris
Cl (
pH 8
.0) b
uffe
r.
62
2.3.8 Restriction Digestion
Restriction reactions were carried out in a total reaction volume of
50 µl with 100 to 1000 ng of DNA, appropriate volume of restriction enzymes
and 1X reaction buffer. The reaction mixture was incubated at 37 ºC for 2 to 3
h. Restriction enzymes were inactivated by heating the mixture at 65 ºC for 20
min. For double digestion, compatible buffers were used for optimal activity
of both enzymes. The digested DNA was further purified using gel extraction
method or spin column method before ligation.
2.3.9 Ligation
Ligation was performed using T4 DNA ligase in reaction volume of
10 µl with appropriate ratios of insert and vector, 1 µl ligase and 2 µl 10X
ligation buffer. The vector/insert ratios were varied from 1:4 to 1:6 for
ligation. The reaction mixture was incubated at 16 ºC for 16 h.
2.3.10 Transformation
2.3.10.1 Transformation of E. coli Strains by Heat Shock
Chemically competent cells of E. coli strains were prepared using
the calcium chloride method (Sambrook et al 1989). Single colony was
inoculated into 4 ml LB broth for overnight, at 37 ºC with shaking at 180 rpm.
The overnight grown culture (250 µl) was used to inoculate 25 ml of LB broth
and grown until the OD600 reached 0.4 to 0.6. The culture was chilled on ice
for 10 min and then harvested by centrifuging at 7500 rpm for 10 min at 4 ºC.
The pellet was gently resuspended in 6 ml of ice cold 0.1M calcium chloride,
incubated in ice for 15 min and centrifuged at 3500 rpm for 20 min at 4 ºC.
The resultant pellet was resuspended in 12 ml of ice cold 0.1M calcium
choloride and kept on ice for 30 min. The cells were centrifuged at 3500 rpm
for 10 min at 4 ºC and the pellet was further resuspended in 500 µl of 0.1M
CaCl2 and stored at 4 ºC for one day as 100 µl aliquots.
63
To 100 µl of competent cells, 5 µl of ligation mixture or plasmid
DNA was added and gently mixed. The mixture was kept at 4 ºC for 30 min
and then subjected to heat shock at 42 ºC for 90 sec in a water bath. The cells
were then immediately chilled on ice for 5 min and 900 µl of LB media was
added. The cells were revived for 1 h at 37 ºC with shaking. The cells were
further plated onto LB agar containing appropriate antibiotics and incubated
at 37 ºC overnight for appearance of colonies.
2.3.10.2 Electrotransformation of L. reuteri
The electrocompetent cells were prepared as described by Berthier
et al (1996). In brief, 100 mL of MRS broth was inoculated with overnight
grown L. reuteri cells to an initial OD600 of 0.35 and incubated at 37 ºC (static
culture). At an OD600 of 0.6, the cells were chilled on ice and harvested,
washed once with ice-cold 10 mM MgCl2, and once with 10 mL of a solution
containing 0.5 M sucrose and 10% glycerol. Finally, cells were resuspended
in 1% of the same solution, aliquoted into eppendorfs and were either used
immediately or stored at -80 ºC upto one month. 5 µl of ligation mixture was
added to 40 µl of electrocompetent L. reuteri cells and transferred to a BTX
electroporator. 460 µL of MRS broth containing 80 mM MgCl2 was added to
the electroporated cells and transferred to an eppendorf and incubated at 37 ºC
for 2 h. The cells were plated on MRS agar containing the required antibiotic
and incubated for 24 - 36 h at 37 ºC until visible colonies were observed.
Electroporation conditions were:
Resistance 800 Ohm
Capacitance 25 µF
Voltage 1.5 kV
Time constant obtained was 11 – 13 milliseconds.
64
2.3.11 Screening of Recombinants by Lysate PCR
Individual transformed colonies were patched on LB agar plate
containing appropriate antibiotic and grown at 37 ºC overnight. A small
portion of patched colony was picked using sterile inoculation loop. The
picked cells were resuspended in 50 µl TrisCl buffer, and washed once.
Further lysis was performed by boiling the cells for 15 min in a water bath.
The lysed cells were centrifuged and 1.0 µl of supernatant was used as
template in 9 µl PCR mix containing forward, reverse primer and 5 µl of
2 x PCR master mixes. Polymerase chain reaction (PCR) was performed in
Eppendorf thermocycler.
2.4 ANALYTICAL METHODS
2.4.1 Agarose Gel Electrophoresis
An agarose gel of required strength (0.7 to 1.2%) was prepared
depending on the size of the DNA fragments to be separated. The gel is
created by suspending the desired quantity of dry agarose in 0.5X TBE buffer
(89 mM Tris, 89 mM Boric acid, 2 mM EDTA, pH 8.3), boiling until the
solution becomes clear, cooling it to about 55 ºC, adding ethidium bromide
(0.5 µg/ml) and then pouring it into a casting tray fitted with sample comb.
The gel was allowed to solidify at room temperature and then the comb was
removed. Further, the gel was placed horizontally in electrophoresis tank
containing TBE (0.5X) buffer. DNA samples were mixed with gel loading
buffer (0.21% Bromophenol Blue, 0.21% Xylene Cyanol FF, 0.2 M EDTA,
pH 8.0 and 50 % glycerol) and loaded onto wells. Bromophenol blue and
Xylene Cyanol dye fronts migrate through agarose gel along with double
stranded DNA fragments of 300 and 4000 bp respectively and thus were used
to mark the migration of DNA during electrophoresis. The voltage was set to
100 to 150 V and electrophoresis was performed at room temperature until
65
Xylene Cyanol reached bottom of the gel. The DNA bands were then
visualized under UV illumination using gel documentation system (Gel Doc
XR+ Imaging System, Biorad).
A similar procedure was adopted for preparing the agarose gel for
RNA analysis, by non-denaturing electrophoresis. 5 µl of RNA samples were
mixed with 5 µl of 2X RNA loading buffer (Qiagen) and heated at 70 ºC for
10 min and snap cooled on ice for 2 min to avoid secondary structure
formation. The sample was then loaded onto agarose gel and electrophoresis
performed at 100 V.
2.4.2 Quantitation of DNA and RNA
The concentration of DNA and RNA can be determined
spectrophotometrically by absorption in ultraviolet range. The readings were
taken at 260 nm and 280 nm. The OD at 260 nm allows calculation of the
concentration of nucleic acid in the sample. An OD260 of 1 corresponds to 50
g/ml for double stranded DNA and 40 g/ml for RNA. The ratio of
absorbance at 260 nm and 280 nm is used to assess the purity of DNA and
RNA. A ratio of ~1.8 is generally accepted as pure for DNA and a ratio of
~2.0 is generally accepted as pure for RNA. If the ratio is appreciably lower
in either case, it may indicate the presence of protein, phenol or other
contaminants that absorb strongly at or near 280 nm.
2.4.3 Cell Disruption by Enzymatic Method
L. reuteri cells were lysed enzymatically, using CelLytic B Plus Kit
(Sigma, USA), as per the manufacturer’s protocol. Briefly, cell pellet from
3 ml culture was resuspended in 1.5 ml of CelLytic B Plus working solution
(1 ml of cell lytic B, 80 l of lysozyme, 20 l of protease inhibitors and 100 U
of benzonase), vortexed briefly and incubated at 22 ºC for 30 min. The sample
66
turned transparent upon cell lysis and release of proteins. An aliquot of lysate
was directly mixed with appropriate volume of SDS sample buffer and
processed for protein analysis by SDS-PAGE. Another aliquot was
centrifuged at 13,000 rpm for 10 min at 4 ºC, to remove any insoluble
material. The soluble fraction was carefully removed from cell debris and
used for enzyme assays.
2.4.4 Total Protein Estimation
Protein concentration was determined with Bio-Rad protein micro
assay based on Bradford method. The dye-binding assay is based on
differential colour change of dye in response to various concentration of
protein (Bradford 1976). The absorbance maximum for an acidic solution of
Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding
to proteins. Each time the assay was performed, a standard curve was
prepared using BSA (Bovine Serum Albumin) as protein standard. 10 µl of
desired concentration of BSA was mixed with 190 µl of dye reagent and
mixed well. The protein concentrations were quantified in the microplate
format, as recommended by the assay kit and absorbance readings were
measured at 595 nm using ELISA reader (Labsystems Multiskan MS).
2.4.5 SDS-PAGE Analysis
Proteins present in sample were analysed by SDS-PAGE according
to the method of Laemmli (1970). The protein sample was mixed with sample
solubilisation buffer (SSB) and boiled to denature proteins for 10 min.
Subsequently, 5 to 15 µl (7 to 15 µg of protein) was loaded on to the gel. The
composition of the acrylamide gels (separating and stacking) is given in Table
2.5. The electrophoresis was started at 70 V until the bromophenol blue
reached the top of separating gel, then increased to 150 V. The electrophoretic
run was completed when the dye reached bottom of the gel.
67
Table 2.5 Components used in SDS-gel prepartion
Separating gel Stacking gel Tris/Hcl, pH 8.8 3.3 ml 3.4 ml
Tris/Hcl, pH 6.8 4.0 ml 830 µl
30 % Acrylamide/Bisacrylamide 2.5 ml 630 µl
10 % SDS 100 µl 50 µl
5 % TEMED 100 µl 50 µl
10 % APS (freshly prepared) 4 µl 5 µl
Acrylamide stock solution
Acrylamide - 29.2 g
Bisacrylamide - 0.8 g
Dissolve in distilled water to a final volume of 100ml
Electrode buffer (10X)
Tris Hcl - 0.25 M
Glycine - 1.92 M
SDS - 10 % (W/V)
Distilled water - upto 500 ml
Sample Solubilization Buffer (SSB, 4X)
Tris Hcl (pH 6.8) - 0.5 M - 1.0 ml
SDS (W/V) - 10 % - 0.8 ml
Glycerol - 40 % - 4.0 ml
- mercaptoethanol - 20 % - 2.0 ml
Bromophenol blue - 0.5 % - 50 µl
Distilled water - upto 10 ml
68
2.4.6 Coomassie Blue Staining
After electrophoresis, the gel was stained with coomassie brilliant
blue R-250 in a clean glass or a plastic container. Staining solution was added
approximately 5 times the gel volume and placed in a shaker for 4 to 14 h.
Stainer was saved for reuse and the gel was subsequently destained using
destainer.
Staining solution for Coomassie blue staining
Coomassie Blue R-250 0.3 g
Methanol 80 ml
Glacial Acetic acid 20 ml
Distilled water 100 ml
Destaining solution for Coomassie blue staining
Glacial Acetic acid 10 ml
Methanol 30 ml
Distilled water upto 100 ml
2.4.7 Biomass Analysis
Cell growth was monitored by measuring OD600 in UV-VIS
spectrophotometer (Hitachi U-1900). Cell dry weight (CDW) was calculated
from a predetermined relationship between CDW and optical density (1 OD600
corresponded to 0.33 g/l CDW and 0.3 g/L CDW for L. reuteri and E. coli
strains respectively).
69
2.4.8 HPLC Analysis
Concentrations of glucose, glycerol, 1,3-PD, ethanol, lactate, 3-
HPA and acetate in the culture broth were determined using a HPLC
(Shimadzu LC-10Avp) that was equipped with a refractive index detector
(RID) and an aminex HPX-87H column (300 x 78 mm, Bio-Rad, USA).
Resin-based HPLC columns like aminex, use multiple mechanisms of ion
exclusion, ion exchange, ligand exchange, size exclusion, reversed phase, and
normal phase partitioning to separate compounds.
Samples were filtered through 0.2µm PVDF (Polyvinylidene
fluoride) membranes before HPLC analysis and 20 µL of the filtered sample
was injected into sample injection port. The following conditions were
maintained during HPLC
Mobile Phase : acetonitrile and water in a ratio of 35:65
in 5 mM H2SO4,
Column temperature : 30 ºC
RID temperature : 50 ºC
Flow rate : 0.4 mL/min
Total time : 30 min
Standard graphs were made by plotting concentration versus area
obtained in the chromatograph. The standard graph equations were further
used to find out unknown concentrations of compounds in the samples.
3-HPA standard was synthesized in the lab using resting cells of L. reuteri
ATCC 55730 as explained in section 2.4.9.
70
2.4.9 Synthesis and Quantitation of 3-HPA
3-HPA was produced as described previously (Luthi-Peng et al
2002a, Spinler et al 2008). Briefly, the protocol for 3-HPA synthesis and
quantitation is as follows:
L. reuteri was cultured in 100 mL MRS broth, incubated
anaerobically at 37 °C for 24 h. The anaerobic condition was
maintained by sparging with nitrogen.
At the end of 24h, the culture was centrifuged and the pellet
washed with 50 mM sodium phosphate buffer (pH 7.4).
The cells were resuspended in 250 mM glycerol to a
concentration of ~1.5 x 1010 cells/mL and incubated
anaerobically at 37 °C for 2 h.
After the 2 h incubation, the culture was pelleted and the 3-
HPA-containing supernatant was collected and filter-sterilized
using a 0.22 µm filter and the filtrate used for HPLC analysis.
Quantitation of 3-HPA was done by HPLC, as described by Spinler
et al (2008). In short, the conversion of glycerol to 3-HPA occurs at a 1:1
ratio (Talarico et al 1988), and the concentration of 3-HPA in each sample
was determined by subtracting the remaining amount of glycerol from the
HPLC-determined concentration of the starting material.
2.4.10 Enzyme Assays
L. reuteri cells were grown on MRS medium containing 111 mM
glucose and 278 mM glycerol, in 15 ml static vials fitted with plastic screw
caps. Erythromycin (5 µg/ml) was added to the medium used for culturing the
71
recombinant strain. Cells were cultivated in batch mode at 37 ºC. Production
of carbon dioxide in the culture created and ensured aerobiosis. The
recombinant culture was induced with SppIP at an OD600 of 0.9 - 1.0. Samples
were taken at logarithmic phase to analyse enzyme activities.
1,3-propanediol oxidoreductase activity was measured
spectrophotometrically by following the initial rate of substrate-dependent
NADH formation at 340 nm ( = 6220 M-1cm-1) by a method adapted from the
work of Heyndrickx et al (1991). The assay mixture contained 100 mM 1,3-
PD, 10 mM NAD+, 30 mM ammonium sulfate, 100 mM Tris buffer (pH 9.0),
2 mM dithiothreitol and the whole cell lysate in a final volume of 1 ml.
The activity of NADP-dependent alcohol dehydrogenase (YqhD)
was also determined spectrophotometrically at 340 nm by the initial rate of
substrate-dependent NADPH increase ( = 6.22 mM-1cm-1). This method was
derived from the method described by Tang et al (2009). The assay mixture
contained 30 mM ammonium sulfate, 100 mM potassium carbonate (pH 9.0),
2 mM NADP, 100 mM 1,3-PD and the whole cell lysate in a final volume of
1 ml. One unit of enzyme activity is defined as the amount of enzyme that
catalyzes the conversion of 1 mol of substrate per min under the specified
conditions. Specific enzyme activity was expressed as units per mg protein.
All assays were performed in duplicates, and reported values are the averages
of two independent experiments.
2.4.11 Quantitative Realtime PCR Analysis
RNA integrity was examined by non-denaturating gel
electrophoresis. A260 and A260/A280 measurements using nanodrop
(Thermo Scientific, USA), were used to estimate the purity and concentration
of the RNA samples.
72
M-MuLV reverse transcriptase was used for cDNA synthesis. The
reaction mixture contained 1 g of RNA, 0.5 mM dNTPs, 0.5 g primers
(random hexamers), 100 U of M-MuLV, made up to a total volume of 60.0 l
with RNAse-free water. The reaction mixture was incubated at 37 °C for
60 min. The reverse transcriptase was then inactivated by heating the reaction
mixture at 65 °C for 10 min. The cDNA was diluted 3 times and stored at
-20 °C until use.
Quantitative real-time polymerase chain reaction (PCR) was
performed using Power SYBR Green PCR master mix (Applied Biosystems,
USA), in HT Fast Realtime PCR, model No 7900 (Applied Biosystems,
USA). The list of oligonucleotides used for qRT-PCR studies are listed in
Table 2.6. PCR was carried out using 96-well plate and the composition of
each reaction is as follows:
Forward primer 1 l (5 pmol)
Reverse primer 1 l (5 pmol)
cDNA 1 l (50 ng)
2X SYBR Green mix 10 l
Sterile water upto 20 l
The reaction mixture was incubated at 95 °C for 10 min, followed
by 40 cycles of denaturation (95 °C) for 15 s, annealing (50 °C) 2 min and
extension of 1 min at 60 °C. The reactions were performed in triplicates for
each gene.
The RQ manager software (Applied Biosystems, USA) was used to
calculate CT values for each gene amplification. The 16S rRNA gene that
codes for 16S ribosomal RNA was used as reference gene. It was used to
73
normalize the amount of cDNA used for each reaction. CT was calculated as
the difference between CT of the gene of interest and CT of the reference gene.
CT was calculated as difference between the delta CT of a gene in the
recombinant strain and CTof the same gene in the control strain at mid-log
phase. In the case of yqhD, CT was calculated for the induced recombinant
strain, with the uninduced strain as reference. The fold change was calculated
using the formula 2 CT (Livak & Schmittgen 2001).
Table 2.6 List of primers used for qRT-PCR analysis
Primer name Primer sequence 5’ to 3’
13pdor (Forward) CGATCCAATGTTGATGCTTGAT
13pdor (Reverse) CATTGACTATCAGTAATTGGGT
yqhD (Forward) GTGTGCTGACGCTGCCA
yqhD (Reverse) GGATCGAGCACGGCAAATAC
pgl (Forward) TCCTCAGAACAGAGGCTTGG
pgl (Reverse) GCAATGGAATTCTCCAAAGCA
fba (Forward) GGTGACTATGATGCAGCGA
fba (Reverse) CAATCTCTTTGGTTTTGGCA
aad (Forward) GCCATCGCTGATATGTGT
aad (Reverse) GTGAAGTGGCCTTATACATTG
ackA (Forward) TGTTGGTCACCGTATCTCTC
ackA (Reverse) AGCCTTTATTCCCGCTAAACCA
ackA2 (Forward) GCATTGTACAGTGTGCCTTA
ackA2 (Reverse) GCACCTGCTCCAATATGCAT
16S rRNA Hufner et al (2008)
74
2.5 STRATEGY FOR CLONING OF YQHD GENE IN
LACTOBACILLUS REUTERI
In order to obtain controlled, high-level expression of yqhD in
L. reuteri, the E. coli – lactobacillus shuttle expression vectors, pSIP 409 and
pSIP 411, were chosen. Both the vectors are characterized by the presence of
certain common features including:
A strong and sakacin-inducible promoter (PorfX)
Gene for erythromycin (ermL ) selection marker that functions
in both E. coli and lactic acid bacteria and
A broad host range and high copy number (pSH71) (de Vos,
1987) as in pSIP 411 or a narrow-host-range Lactobacillus
replicon from plasmid p256 (Sørvig et al 2005) as in pSIP 409.
The 1.163 kb yqhD gene fragment (GenBank accession number
NC010498), was amplified from the chromosomal DNA of E. coli K-12
MG1655 using the primers yqhDF and yqhDR (Table 2.7). PCR conditions
employed were:
1. Initial denaturation 98 ºC 10 sec
2. Denaturation 98 ºC 3 sec
3. Annealing 65 ºC 5 sec
4. Extension 72 ºC 20 sec (1 kb/15 sec)
5. Final extension 72 ºC 1 min
24 cycles from step 2 to 4
75
The amplicon was cloned into TA vector and transformed into
E. coli DH5 to generate the recombinant plasmid pHR1. Further, the yqhD
gene was sub-cloned from pHR1 into NcoI/XhoI site of pSIP411 and pSIP
409, with E. coli EC1000 as the cloning host, to yield constructs pHR2 and
pHR3 respectively. However, while pHR2 could be easily electroporated into
L. reuteri ATCC 55730, to generate the recombinant strain L. reuteri HR2,
the pHR3 plasmid could not be transformed due to the incompatibility of its
256rep replicon with the host. The clones were screened by lysate PCR using
the primer pair PorfXF and yqhDR (Table 2.7). The strategy used for
generating L. reuteri HR2 is depicted in Figure 2.1.
Figure 2.1 Strategy adopted for cloning yqhD gene in L. reuteri ATCC
55730
76
Table 2.7 Primers and peptide sequences used in the study
Primer name Primer sequencea
yqhDF (Forward) 5’-CATG CCATGGACAACAACTTTAATCTGCACACC-3'
yqhDR (Reverse) 5‘-CCG CTCGAG TTAGCGGGCGGCTTC-3’
PorfXF (Forward) SppIP
5’-TGAAAATTGATATTAGCG-3’ MAGNSSNFIHKIKQIFTHR
a The restriction sites in the primers NcoI (forward) and XhoI (reverse) have been underlined
2.6 METABOLIC FLUX ANALYSIS
In order to perform flux analysis, L. reuteri strains were grown on
semi-defined medium in 250 ml static flasks fitted with plastic screw caps.
They were inoculated with early log phase culture to obtain an initial OD600 of
0.2-0.3. They were cultivated in batch mode at 37 ºC for 7 h with an initial pH
of 6.5. Production of carbon dioxide in the culture created and ensured
anaerobiosis. The recombinant culture was grown in two different conditions
– uninduced and induced with SppIP at an OD600 of 0.6. Aliquots were
removed periodically to track growth and analyse metabolite profiles.
Experiments were carried out in duplicate and the mean values were estimated
together with the standard deviations for OD600, specific growth rate and
fermentation products.
Metabolic flux analysis is based on the pseudo-steady-state
assumption for intracellular metabolites, which implies that there is no
accumulation of any intermediates. Metabolic flux analysis for glucose and
glycerol – grown cells consisted of the following fluxes and unknown
intermediates: the glycolytic pathway consisted of twelve fluxes (v1 to v12)
and nine intermediates (Figure 2.2); the glycerol pathway comprised of three
fluxes (v13 to v15) and one intermediate (Figure 2.2). The degrees of freedom
77
for the glucose and glycerol pathways are 3 and 2 respectively. The system
becomes classically over determined as number of measured fluxes include
four in glycolytic and three in glycerol dissimilation pathways. These fluxes
were calculated from substrate utilization and product formation data using a
stoichiometric model of glucose and glycerol metabolism (Figure 2.2). The
metabolic fluxes through phosphoketolase (PKP), Embden-Meyerhof (EMP)
and glycerol dissimilation pathways were estimated for the period of
maximum growth rate as described by Årsköld et al. (2008). The fluxes in the
stoichiometric model, were derived from the relationship Qx = max * Yx,
where Qx is flux of the specific metabolite x (expressed as mol of x / mol
biomass-h), max represents maximum specific growth rate (h-1) and Yx
represents the specific metabolite production (mol of x / mol biomass). The
carbon balances were determined for control, uninduced and induced
recombinant L. reuteri strains, based on the total amount of carbon-containing
products and biomass formed (in C-mol) and the amount of glucose and
glycerol consumed (in C-mol). Carbon balances for glucose estimated during
the exponential growth phase in control and uninduced cultures were found to
close on C-mol basis at ~103%. However, the fluxes could not be analyzed
for the induced recombinant strain since carbon balance closure could not be
attained (data not shown). Carbon dioxide production was calculated from the
stoichiometric relationship with acetate and ethanol production (Arskold et al
2008).
To understand the influence of the heterologous alcohol
dehydrogenase on carbon flux, flux partitioning at key branch points, was
examined as previously described (Sridhar & Eiteman 2001). To determine
the flux-partitioning at a specific node, the carbon flux entering that node was
scaled to 100. Each scaled flux exiting the specific node therefore represents
the fraction of the incoming carbon flux exiting by that route.
78
Figure 2.2 The glycolytic and glycerol dissimilation pathways and
corresponding in vivo fluxes in L. reuteri ATCC 55730 The fluxes in glycoltic pathways are represented by v1 to v12 and fluxes in glycerol pathway by v13 to v15. Abbreviations: G6P glucose-6-phosphate, 6PG 6-phosphogluconate, R5P ribulose-5-phosphate, X5P xylulose-5-phosphate, AcP acetyl phosphate, AcCoA acetyl-CoA, F6P fructose-6-phosphate, FBP, fructose-1,6-bisphosphate, DHAP dihydroxyacetone phosphate, GAP glyceraldehyde-3-phosphate, Pyr pyruvate, 3-HPA 3-hydroxypropionaldehyde, GDHtglycerol dehydratase, 1,3-PDOR 1,3-propanediol oxidoreductase, YqhD E. coli alcohol dehydrogenase
79
2.6.1 Analysis of Glycerol Metabolism in L. reuteri ATCC 55730
using 13C- NMR Studies
For the labeling experiment, the following protocol was followed:
i. Inoculated 2 ml MRS broth with 10% of overnight grown L.
reuteri culture. Incubated at 37 °C for 1-2 h, until the culture
entered the log phase (OD600 of 0.9 to 1.0 obtained)
ii. 500 µl of the log phase culture was used to inoculate 5 ml of
MRS broth containing 13C3-labeled glycerol (130 mg).
Incubated the culture at 37 °C for 10 - 12 h, until the culture
reached late stationary phase (OD600 of 4.5 reached).
iii. Culture harvested and centrifuged to separate the fermentation
broth containing extracellular metabolites.
iv. The supernatant was concentrated by lyophilisation,
redissolved in 500 l of deuterated water (Sigma-Aldrich, St.
Louis, MO) and used for the identification of extracellular
metabolites.
13C-NMR spectra were recorded at 125.77 MHz on a Bruker AV III
500 MHz FT spectrometer with a broadband gradient probe head (5mm) at
room temperature. Signals for glycerol and 1,3-propanediol were assigned by
comparison with previously published chemical shifts (Spectral Database for
Organic Compounds, Japan) (A 1.2 and 1.3).
2.7 BIOREACTOR STUDIES
2.7.1 Batch Fermentation
To study the effect of glucose and glycerol concentration on growth
and metabolite formation in L. reuteri ATCC 55730, co-fermentation was
80
performed in batch mode with two different ratios of glucose and glycerol, 0.4
and 0.5, in MRS medium. The inoculum for the batch reactor was grown in
150 mL MRS broth at 37 ºC until an OD600 of 0.8 – 1.0 was reached. The seed
was then inoculated into a 2 L fermentor (KLF 2000 – Bioengineering AG,
Switzerland) filled with 1.2 L MRS medium containing erythromycin and
glycerol (278 mM or 217 mM). Fermentation was carried out at 37 ºC and
250 rpm, in an anaerobic condition. The pH was maintained at 5.5 by the
addition of 1.5 M NaOH or 1.5 M H3PO4 (El-Ziney et al. 1998). The
anaerobic condition was established by flushing with sterile nitrogen.
Similar batch bioreactor studies involving the recombinant L.
reuteri strain were carried out in MRS medium with erythromycin. A glucose
to glycerol ratio of 0.4 was used. At 0.8 OD600, the recombinant culture was
induced with 50 ng/mL of sakacin P induction peptide (SppIP). Samples were
removed periodically for determining OD600. The culture pellet and
supernatant were stored at – 20 ºC, to be used later for protein and metabolite
analyses respectively.
Cultures for qRT-PCR studies, were grown in a 7.5 L fermentor
(New Brunswick Scientific, BIOFLO 415 Fermentor, USA) filled with 3 L
MRS medium containing glycerol (278 mM). Rest of the procedure was
similar to that of the batch fermentation described earlier. The samples were
removed at mid-log and late-log phase for qRT-PCR analysis.
2.7.2 Fed-batch Fermentation
Fed-batch fermentation was performed with L. reuteri ATCC
55730 strain, in a 7-L fermentor (BioFlo 415-New Brunswick Scientific,
USA) containing 3-L MRS medium at 37 ºC, 250 rpm and pH 5.5 (controlled
by adding 1.5 M NaOH or 1.5 M H3PO4) supplemented with 1.84 g/L
(20 mM) glycerol, under non-aerated conditions. The pre-culture was grown
81
in 300ml MRS broth at 37 ºC until an OD600 of 0.8-1.0 was reached and was
inoculated into fermenter. Two kinds of feed were given – Feed solution I
contained only glucose and glycerol, whereas feed solution II contained
limiting nutrients (Table 2.8). Two types of feed solution I (A and B) were
used in this study. Feed Solution I (A) containing low concentration of
glycerol, was used to primarily improve growth and prevent the accumulation
of toxic 3-HPA. Since 1,3-PD is growth-associated, feed Solution I (B)
containing higher glycerol concentrations, was given when an OD600 of ~9.5
was reached, to improve cell density as well as 1,3-PD formation.
Table 2.8 Composition of feed solutions used in fed-batch study
Type of feed Composition
Feed solution I (A) Glucose - 720 g/l (4 M)
Glycerol - 73.6 g/l (0.8 M)
Feed solution I (B) Glucose - 362.25 g/l (2.0 M)
Glycerol - 287.5g/l (3.12 M)
Feed solution II Beef extract - 200 g/l
peptone - 20 g/l
Yeast extract - 20 g/l
At glucose concentrations below 5 g/L (27.7 mM), 42 g of Feed
Solution I (A) and 60g of Feed solution II were added to the reactor medium.
For second phase feed, 114.78 g of Feed Solution I (B) and 60g of Feed
solution II were added simultaneously into the reactor. Samples were
collected at regular intervals for monitoring OD600. The samples were also
centrifuged and the supernatant stored at – 20 ºC, to be used later for
metabolite analyses.
82
2.8 1,3-PROPANEDIOL TOLERANCE STUDIES IN L. REUTERI
The inhibitory effect of 1,3-propanediol on growth of L. reuteri
was studied under batch conditions in 50 ml bottles fitted with plastic screw
caps. Different concentrations of 1,3-PD – 0, 50,100 and 150 g/L, were added
to the growth medium (MRS broth), either before inoculation or when the
culture reached early exponential phase (OD600 1.0) and incubated at 37 °C
for 24 h. Growth was monitored by measuring OD600 at regular intervals. The
absorbance values were then converted to Cell dry weight (CDW) values as
explained in 2.4.7.
2.9 BY-PRODUCT INHIBITION ANALYSIS
Formation of by-products, lactate and ethanol, was curtailed using
inhibitors. Log phase cultures of L. reuteri grown in MRS were exposed to
varying concentrations of lactate inhibitors, sodium iodoacetate and oxalic
acid for up to 4 h and their growth and metabolite production analysed by
HPLC. To inhibit ethanol formation, cultures of native and engineered strains
of L. reuteri in latent (before inoculation) and log phase in MRS, were treated
with 15mM benzaldehyde, an ethanol inhibitor (Shone et al 1981) and
incubated at 37 °C for 24 h. Growth was monitored by measuring OD600. The
concentration of acetate, lactate, ethanol and 1,3-PD in the culture supernatant
were determined by HPLC.
10% of the overnight grown L. reuteri cultures were transferred to
15 ml vials containing MRS with 111mM glucose and 278mM glycerol and
was incubated at 37 °C till early log phase of 0.8 -1.0 was reached. Varying
concentration of lactate and ethanol inhibitors were added to the culture at log
phase and samples were collected at different time points to monitor cell
growth and metabolite concentrations.
83
2.10 BIOINFORMATIC ANALYSIS
To determine if the sequence of the redox-sensing global regulatory
protein Rex and its DNA-binding sites, are present in the genome of
Lactobacillus reuteri ATCC 55730, bioinformatic analysis was adopted.
However, genome of L. reuteri ATCC 55730 strain is not manually curated
for the presence of Rex repressor and its binding sites. Therefore relevant
information from another strain, L. reuteri JCM 1112, that is available at
RegPrecise, an online database of manually curated inferences of regulons
(Novichkov et al 2010), was exploited. The amino acid sequence of Rex
protein from L. reuteri JCM 1112 strain (UniProt id: B2G5X5) was blasted
against the whole genome of L. reuteri ATCC 55730 to identify the
corresponding putative Rex sequence. In the rex regulon of JCM 1112 strain,
Rex protein was found to bind upstream of the following five genes, namely,
rex (Redox-sensitive transcriptional regulator Rex), gldA (Glycerol
dehydrogenase), dhaT (1,3-propanediol dehydrogenase), noxE (NADH
oxidase), adhE (Alcohol dehydrogenase, EC 1.1.1.1; Acetaldehyde
dehydrogenase, EC 1.2.1.10). The Rex-binding sites upstream of the 5 genes
in JCM 1112 strain were used as queries and blasted against the whole
genome of ATCC 55730 strain, to identify the presence of similar putative
Rex-binding sites.
2.11 SIMPLE UNSTRUCTURED KINETIC MODEL
A modified version of the kinetic model proposed by Tobajas et al
(2008) has been adapted to describe the evolution of biomass, consumption of
substrates and formation of products at various time points, in glucose-
glycerol co-ferementation in L. reuteri. The kinetic model was used by the
authors to describe the behaviour of L. reuteri PRO 137 strain during
co-fermentation in batch mode. In the present study, the modified model has
been utilized for describing the behaviour of native and recombinant
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(L. reuteri HR2) strains of L. reuteri ATCC 55730. The simple unstructured
model developed by my colleague, Ms. Geethalakshmi, considers the mass
balances for eight state variables such as biomass (X), glucose (S), glycerol
(G), acetate (A), lactate (L), ethanol (E), 3-HPA (R) and 1,3-propanediol (P)
(Equations 2.1 – 2.8) with eight parameters , viz., k1, k2, k3, k4, YXS, YLX, YEX and
YAX. Essentially all the mass balance equations have been adopted from the
model proposed by Tobajas et al (2009) except the ones describing the
production rate of 1,3-PD and glycerol, which have been slightly altered.
The equation used to describe the growth of L. reuteri not only
considers biomass evolution and substrate concentrations, but also the
inhibitory influence of 3-HPA accumulation. This is given by:
RkXSk
dtdX
2
1 (2.1)
where 1k represents the growth constant and 2k represents the saturation
constant for 3-HPA.
Since glucose is the limiting substrate for the growth and biomass
formation in L. reuteri, the rate of glucose consumption could be linked to the
yield of biomass on glucose as follows:
/( )XS
dS dX dtdt Y
(2.2)
where YXS denontes the yield of biomass on glucose.
The equations describing the production rate of 1,3-PD and
glycerol are based on the conversion of glycerol to reuterin and its subsequent
reduction to 1,3-PD, as is evident from experimental data in literature (Luthi-
Peng et al 2002a, Talarico et al 1990). The additional direct glycerol
85
bioconversion assumed in the earlier model, was omitted in our study.
Accordingly the equations describing the glycerol consumption rate and 1,3-
PD formation rate have been modified. The rate of glycerol consumption
depends on biomass and reuterin formation as given by the expression:
GkdtdG X3 (2.3)
where 3k is the Kinetic constant for 3-HPA production.
The rate at which reuterin is produced is related to its production by
glycerol dehydration as well as its conversion to 1,3-PD by reduction. Both of
these processes are reflected in the equation:
XRkXGkdtdR
43 - (2.4)
where 4k is the Kinetic constant for 1,3-propanediol production
The equation describing the formation rate of 1,3-PD could be
directly linked to the reduction of reuterin as:
XRkdtdP
4 (2.5)
The rate of production of by-products such as lactate, acetate and
ethanol, could be related to cell growth as expressed by the following rate
equations:
dXdtdL YLX / dt (2.6)
where LXY is the yield of lactate on biomass.
86
dXdtdE YEX / dt (2.7)
where EXY is the yield of ethanol on biomass.
dXdtdA YAX / dt (2.8)
where AXY is the yield of acetate on biomass.
2.11.1 Parameter Estimation
Parameters of the unstructured model have been estimated using
genetic algorithm (GA), an effective stochastic global search algorithm (Park
et al 1997, Roubos et al 1999, Sarkar and Modak 2004), along with a Runge-
Kutta integration method. Experimental data of the batch experiments have
been interpolated for accurate parameter estimation. The parameters have
been estimated by minimizing the objective function as given in Equation
(2.9), which is the normalized squared error between the estimated and
measured state variables in MATLAB 7.7. The optimization tool box with
Genetic algorithm as a solver has been used to estimate the parameters. The
objective function is defined as,
m
j
n
iij ijeij XXX
1 1
2max
2functionObjective (2.9)
where n is the number of observations (interpolated data)
m is the number of state variables
Xeij represents the estimated state variables
Xij represents the measured state variables and
Xij max is the maximum value of the state variable
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In an attempt to make the model more realistic, the estimated
parameters were constrained by upper and lower boundary values that were
based on batch data and relevant literature (De Vries et al 1970, Pancheniak
et al 2012, Teusink et al 2006, Tobajas et al 2009). The estimated parameters
have been used to simulate the entire profile of the state variables for the two
batch experiments, by solving the above nonlinear differential equations in
MATLAB.