Please, find in this document the measured IPTG distribution profiles of the nine
fermentations induced with IPTG when 20 g DCW·L-1 were achieved.
B
10
10
40
8
8
30
6
20
4
10
2
0
[IPTG]medium (M)
IPTG in medium (mol)
IPTG in medium
DCW
DCW (g·L-1)
A
1
2
3
4
2
0
0
6
0
4
0
1
Time after induction (h)
2
3
4
Time after induction (h)
Figure Error! No text of specified style in document..1 IPTG distribution profiles in
medium in terms of amount (µmol) along with the biomass evolution (A) and in terms
of concentration (µM) (B) after induction of RhuA overexpression with 8 µM IPTG.
Solid line indicates the fitted curve. Fermentation code: FB1
B
120
IPTG intracellular
RhuA
0,08
100
80
0,06
60
0,04
40
0,02
[IPTG]intracel (M)
0,10
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)measured
A
40
30
20
10
20
0,00
0
0
0
1
2
3
0
4
1
2
3
4
Time after induction (h)
Time after induction (h)
Figure Error! No text of specified style in document..2 IPTG distribution profiles inside
the biomass in terms of amount (µmol·g-1DCW) along with the specific RhuA
production (A) and in terms of concentration (µM) (B) after induction of RhuA
overexpression with 8 µM IPTG. Fermentation code: FB1
A
0,14
120
0,12
100
B
70
80
0,08
60
0,06
40
0,04
IPTG intracellular 20
RhuA
0,02
0,00
0
0
1
2
3
Time after induction (h)
4
[IPTG]intracel calc (M)
0,10
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)calc
60
50
40
30
20
10
0
0
1
2
3
4
Time after induction (h)
Figure Error! No text of specified style in document..3 Calculated IPTG distribution
profiles inside the biomass in terms of amount (µmol·g-1DCW) along with the specific
RhuA production (A) and in terms of concentration (µM) (B) after induction of RhuA
overexpression with 8 µM IPTG. Fermentation code: FB1
35
6
30
4
25
2
20
0
15
0
1
2
3
4
14
12
40
8
DCW (g·L-1)
IPTG in medium (mol)
B
45
IPTG in medium
DCW
10
[IPTG]medium (M)
A
10
8
6
4
2
0
5
0
Time after induction (h)
0,6
D 300
180
0,08
180
160
140
140
0,06
5
0,4
120
120
100
100
0,3
0,04
80
0,2
60
80
60
0,02
40
40
IPTG intracellular
RhuA
0,1
250
[IPTG]intracel (M)
0,5
20
200
150
100
50
20
0,0
0
0,00
00
11
22
33
44
55
0
0
0
Time
Timeafter
afterinduction
induction(h)
(h)
E
2
3
4
Time after induction (h)
160
RhuA (mg·g-1DCW)
-1 -1
mol
DCW)
IPTG·g
mol
DCW)
IPTG·g
calc
C
1
0,08
F
180
1
2
3
4
5
Time after induction (h)
40
120
100
0,04
80
60
0,02
40
IPTG intracellular
RhuA
[IPTG]intracel calc (M)
140
0,06
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)calc
160
30
20
10
20
0,00
0
0
1
2
3
Time after induction (h)
4
5
0
0
1
2
3
4
5
Time after induction (h)
Figure Error! No text of specified style in document..4 IPTG distribution profiles along
time after induction of RhuA overexpression with IPTG 10 µM. (A) in medium in terms
of amount (µmol) along with the biomass evolution, (B) in terms of concentration (µM)
and the obtained fitted curve, (C) inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production, (D) inside the biomass in terms of
concentration (µM) and, (E) calculated inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production and (F) calculated inside the biomass
in terms of concentration (µM). Fermentation code: FB2
A
20
B
40
25
10
25
5
IPTG in medium
DCW
0
1
2
3
4
5
15
0
0
5
80
0,04
60
0,03
40
0,02
20
IPTG intracellular
RhuA
0,00
RhuA (mg·g-1DCW)
(mol IPTG·g-1DCW)
0,05
1
2
3
4
2
4040
0,10
0,02
IPTG intracellular
RhuA
0,00
0,00
2020
00
44
Timeafter
afterinduction
induction
Time
(h)(h)
55
[IPTG]intracel calc (M)
0,03
RhuA (mg·g-1DCW)
6060
0,15
F
RhuA (mg·g-1DCW)
(mol IPTG·g DCW)
mol IPTG·g -1DCW)
calc
-1
8080
33
5
4
1
2
3
Time after induction (h)
0,04
22
4
6
0
0,05
0,20
11
5
0
100
100
00
4
8
5
0,25
0,06
0,05
0,01
3
10
Time after induction (h)
E
2
12
0
0
1
Time after induction (h)
D
100
0,01
10
20
Time after induction (h)
C 0,06
15
[IPTG]intracel (M)
0
[IPTG]medium (M)
30
DCW (g·L-1)
IPTG in medium (mol)
20
35
15
100
80
60
40
20
0
0
1
2
3
4
5
Time after induction (h)
Figure Error! No text of specified style in document..5 IPTG distribution profiles along
time after induction of RhuA overexpression with IPTG 20 µM. (A) in medium in terms
of amount (µmol) along with the biomass evolution, (B) in terms of concentration (µM)
and the obtained fitted curve, (C) inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production, (D) inside the biomass in terms of
concentration (µM), (E) calculated inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production and (F) calculated inside the biomass
in terms of concentration (µM). Fermentation code: FB3
A
40
B
34
44
28
36
26
34
24
[IPTG]medium (M)
30
DCW (g·L-1)
IPTG in medium (mol)
32
38
22
32
20
30
42
40
38
36
18
0
1
2
3
4
0
1
Time after induction (h)
0,014
0,05
120
60
80
0,03
0,008
40
60
0,006
0,02
40
0,004
0,01
20
IPTG intracellular
RhuA
0,002
0,00
00
11
22
3 3
4 4
5
RhuA (mg·g DCW)
100
0,04
0,010
5
4
3
2
20
1
0
0
0
0
1
0,03
0,02
0,01
0,04
80
60
0,03
40
0,02
20
IPTG intracellular
RhuA
0,01
120
100
80
60
40
20
0,00
F
140
[IPTG]intracel calc (M)
0,04
100
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)calc
(mol IPTG·g-1DCW)
0,05
0,05
2
3
4
Time after induction (h)
RhuA (mg·g-1DCW)
E
4
7
Time after
after induction
Time
induction(h)(h)
0,06
3
6
80
0,012
0,000
D
[IPTG]intracel (M)
100
140
RhuA (mg·g-1DCW)
0,06
0,016
-1
-1
-1
mol IPTG·g
(mol
DCW)
IPTG·gDCW)
C
2
Time after induction (h)
25
20
15
10
5
0
0
10,00
0
2
1
3
4
2
5
3
Time after induction
Time (h)
after induction (h)
0
4
0
0
1
2
3
4
5
Time after induction (h)
Figure Error! No text of specified style in document..6 IPTG distribution profiles along
time after induction of RhuA overexpression with IPTG 40 µM. (A) in medium in terms
of amount (µmol) along with the biomass evolution, (B) in terms of concentration (µM)
and the obtained fitted curve, (C) inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production, (D) inside the biomass in terms of
concentration (µM), (E) calculated inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production and (F) calculated inside the biomass
in terms of concentration (µM). Fermentation code: FB4
A
B
32
60
40
28
30
26
20
24
[IPTG]medium M
30
DCW (g·L-1)
IPTG in medium (mol)
50
22
10
IPTG in medium
DCW
0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
65
55
50
45
40
20
35
18
30
0,0
3,5
0,5
C
0,7
D
140
0,06
1,5
2,0
2,5
250
0,03
0,02
0,01
120
0,5
80
0,4
60
0,3
40
0,2
0,1
20
IPTG intracellular
RhuA
0,0
0,00
0
0,0
1
0,5
2
1,0
3
1,5
2,0
4
2,5
Time after induction (h)
Time after induction (h)
0
5
3,0
3,5
100
80
60
[IPTG]intracel calc (M)
0,04
0,6
RhuA (mg·g -1DCW)
0,05
RhuA (mg·g-1DCW)
calc
100
(mol IPTG·g -1DCW)
(mol IPTG·g-1DCW)
1,0
Time after induction (h)
Time after induction (h)
200
150
100
40
50
20
0
0,0
0,5
1,0
1,5
2,0
2,5
Time after induction (h)
Figure Error! No text of specified style in document..7 IPTG distribution profiles along
time after induction of RhuA overexpression with IPTG 54 µM. (A) in medium in terms
of amount (µmol) along with the biomass evolution, (B) in terms of concentration (µM)
and the obtained fitted curve, (C) calculated inside the biomass in terms of amount
(µmol·g-1DCW) along with the specific RhuA production and (D) calculated inside the
biomass in terms of concentration (µM). Fermentation code: FB5. Note: Intracellular
samples could not be analyzed and some of the medium samples were missed.
100
40
80
35
60
30
40
25
20
20
120
IPTG in medium
DCW
[IPTG]medium (M)
DCW (g·L-1)
100
15
0,5
1,0
1,5
2,0
2,5
3,0
D
0,12
140
0,10
120
100
0,08
80
IPTG intracel
RhuA
0,06
60
0,04
40
0,02
0,00
1,0
1,5
2,0
2,5
3,0
3
50
30
20
0
3,5
0
1
Time after induction (h)
2
3
Time after induction (h)
E
F
0,8
140
350
300
0,6
100
80
0,4
60
40
0,2
IPTG intracellular
RhuA
20
0,0
0
0,0
0,5
1,0
1,5
2,0
2,5
Time after induction (h)
3,0
3,5
[IPTG]intra calc (M)
120
RhuA (mg·g-1DCW)
(mol IPTG·g-1DCW)calc
2
10
0
0,5
1
Time after induction (h)
40
20
0,0
40
0
Time after induction (h)
C
60
0
3,5
[IPTG]intracel ()
0,0
80
20
0
mol IPTG·g-1DCW)
B
RhuA (mg·g-1DCW)
IPTG in medium (mol)
A
250
200
150
100
50
0
0
1
2
3
Time after induction (h)
Figure Error! No text of specified style in document..8 IPTG distribution profiles along
time after induction of RhuA overexpression with IPTG 100 µM. (A) in medium in
terms of amount (µmol) along with the biomass evolution, (B) in terms of concentration
(µM) and the obtained fitted curve, (C) inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production, (D) inside the biomass in terms of
concentration (µM), (E) calculated inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production and (F) calculated inside the biomass
in terms of concentration (µM). Fermentation code: FB6
B
30
200
26
160
24
140
22
120
[IPTG]medium (M)
28
180
DCW (g·L-1)
IPTG in medium (mol)
A
1,0
1,5
2,0
2,5
150
100
50
18
100
0,5
200
20
IPTG in medium
DCW
0,0
250
0,0
3,0
0,5
Time after induction (h)
C
D
0,20
120
100
0,14
0,12
80
0,10
60
0,08
0,06
40
[IPTG]intracel (M)
0,16
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)
1,5
2,0
2,5
3,0
80
140
0,18
60
40
20
0,04
20
IPTG intracellular
RhuA
0,02
0,00
0
0
0,0
0,5
1,0
1,5
2,0
2,5
0,0
3,0
0,5
1,0
1,5
2,0
2,5
3,0
Time after induction (h)
Time after induction (h)
E
1,0
Time after induction (h)
2,0
140
F
1000
100
80
1,0
60
40
0,5
IPTG intracellular
RhuA
20
0,0
0
0,0
0,5
1,0
1,5
2,0
Time after induction (h)
2,5
3,0
800
[IPTG]intracel calc (M)
1,5
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)calc
120
600
400
200
0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Time after induction (h)
Figure Error! No text of specified style in document..9 IPTG distribution profiles along
time after induction of RhuA overexpression with IPTG 200 µM. (A) in medium in
terms of amount (µmol) along with the biomass evolution, (B) in terms of concentration
(µM) and the obtained fitted curve, (C) inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production, (D) inside the biomass in terms of
concentration (µM), (E) calculated inside the biomass in terms of amount (µmol·g1
DCW) along with the specific RhuA production and (F) calculated inside the biomass
in terms of concentration (µM). Fermentation code: FB7
28
380
26
360
24
340
22
320
460
440
[IPTG]medium (M)
400
B
DCW (g·L-1)
30
IPTG in medium (mol)
A 420
420
400
380
20
IPTG in medium
DCW
360
300
18
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
0,0
Time after induction (h)
C
D
0,4
0,5
1,0
1,5
2,0
2,5
3,0
Time after induction (h)
160
100
60
0,2
40
0,1
[IPTG]intracel (M)
0,3
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)
140
80
1,5
60
40
0
0
1,0
80
20
0,0
0,5
100
20
IPTG intracellular
RhuA
0,0
120
2,0
2,5
3,0
0
3,5
mol IPTG·g-1DCW)calc
2,0
80
1,5
60
1,0
40
0,5
20
IPTG intracellular
RhuA
0,0
0
0,5
1,0
1,5
2,0
2,5
Time after induction (h)
3,0
3,5
F
2
3
1200
1000
[IPTG]intracel calc (M)
100
RhuA (mg·g DCW)
2,5
-1
E
0,0
1
Time after induction (h)
Time after induction (h)
800
600
400
200
0
0
1
2
3
Time after induction (h)
Figure Error! No text of specified style in document..10 IPTG distribution profiles
along time after induction of RhuA overexpression with IPTG 450 µM. (A) in medium
in terms of amount (µmol) along with the biomass evolution, (B) in terms of
concentration (µM) and the obtained fitted curve, (C) inside the biomass in terms of
amount (µmol·g-1DCW) along with the specific RhuA production, (D) inside the
biomass in terms of concentration (µM), (E) calculated inside the biomass in terms of
amount (µmol·g-1DCW) along with the specific RhuA production and (F) calculated
inside the biomass in terms of concentration (µM). Fermentation code: FB8
IPTG in medium (mol)
A 1000
B
34
32
900
1000
26
24
700
22
DCW (g·L-1)
28
[IPTG]medium (M)
30
800
20
600
500
0,0
0,5
1,0
1,5
1100
IPTG in medium
18
DCW
16
2,0
2,5
3,0
900
800
700
600
500
0,0
Time after induction (h)
D
160
1,4
140
1,2
120
IPTG intracellular
RhuA
1,0
100
0,8
80
0,6
60
0,4
40
0,2
20
0,0
[IPTG]intracel (M)
1,6
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)
C
0,5
1,0
1,5
2,0
2,5
1,5
2,0
2,5
3,0
400
300
200
0
3,0
0,0
Time after induction (h)
E
1,0
Time after induction (h)
100
0
0,0
0,5
0,5
1,0
1,5
2,0
2,5
3,0
Time after induction (h)
5
F
160
2500
120
100
3
80
60
2
40
1
20
IPTG intracellular
RhuA
[IPTG]intracel calc (M)
4
RhuA (mg·g-1DCW)
mol IPTG·g-1DCW)calc
140
2000
1500
1000
500
0
0
0
0,0
0,5
1,0
1,5
2,0
Time after induction (h)
2,5
3,0
0
1
2
3
Time after induction (h)
Figure Error! No text of specified style in document..11 IPTG distribution profiles
along time after induction of RhuA overexpression with IPTG 1000 µM. (A) in medium
in terms of amount (µmol) along with the biomass evolution, (B) in terms of
concentration (µM) and the obtained fitted curve, (C) inside the biomass in terms of
amount (µmol·g-1DCW) along with the specific RhuA production, (D) inside the
biomass in terms of concentration (µM), (E) calculated inside the biomass in terms of
amount (µmol·g-1DCW) along with the specific RhuA production and (F) calculated
inside the biomass in terms of concentration (µM). Fermentation code: FB9