¿Podemos diseñar sistemas de expresión que respondan a señales ambientales predeterminadas?...
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¿Podemos diseñar sistemas de expresión que respondan a
señales ambientales predeterminadas?
Reguladores a la carta
The transcriptional regulator XylR
A (receptor)
B interdomain
ATP binding
C (activation) D HTH
DNA binding in Pu y Ps
COOHNH2
211 233
Recognition and binding of aromatic effectors
ATP hydrolysisMultimerizationContacts with 54
R
R
XylR active
B
D
A
C
XylR inactive
Intramolecular repressionSpecific A-C interactions
Binding to A domainRelease of repression
Activation of XylR in response to inducers
R
B
D
A
C
Regulators á la carte: can we change at will the effector specificity of XylR?
The B domain of XylR: a coiled coil?
A C D
B/Q linker
1 211 472233 514 554
CN
566
208 236
(XylR16-1) DP HHHR (XylR3H)
IVDE RYELQTQ VANLRNR LKQYDGQ YYGIG
Native inducer
XylR
BA
C
OH4
phenolF
CH3
7
4-FBA
CH3
Cl
8
4-ClBA
CH3
11
1-methyl
13
biphenyl
CH3-(CH2)6-CH3
14
octane
CH3
CH3
m-xileno
2
5
benzene
NH2
6
aniline
CH3
NO2
9
4-NT
10
naphthalene
CH3
12
2-methyl
CH3
Cl
3-Cl tol
3
Suboptimal inducers
Non-inducers
0
5
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14
BC
MAD16-1, XylR16-1
Inducer compounds
0
5
10MAD1, XylR
BA
cti
vity
Pu
(-
Ga
l, x
10
3)
Effects of 16-1 mutation in the B domain of XylR
A
A
B
D
A
C
Can we change the effectorspecificity of XylR?
R
R
Non naturalinducers
XylRactive
Generation of regulators á la carte
D
AB
C
XylRinactive
Structural prediction
The A domain of XylR
Genetic approach: generation of diversity
1 211
CH3
NO2
D
AB
C
XylR
CH3
NO2
CH3
NO2
toluene m-xylene
2-NT 3-NT 4-NT biphenyl
p-xylene
CH3 CH3
CH3
CH3
CH3
Native inducers
New inducers
Reshaping the effector pocket of the A domain
A
CH3
CH3
generation
of diversity
R
R
1. Preparation of target DNA2. Digestion DNase I (30-300 bp)
3. PCR without oligonucleotides
4. PCR with oligonucleotides
Generation of diversity through mutation-prone shuffling of homologous A domains
XylR A domainDmpR A domain TbuT A domain
Family of similarDNA sequences
Genetic screening
Elimination ofNon productivecombinations
Selection ofnew activecombinations
Shuffled A domains
Pool of A domainvariants
Shuffling of A domains of XylR-like activators
XylR A domainDmpR A domain TbuT A domain
Family of similarDNA sequences
The genetic tricks
• Positive selection
• Negative selection
• Visual screening
• Phenotypical characterisation
Po npt (km)
Po sacB
Po luxAB
Pu lacZ
Cloning vector
pCon918
A C DPr
NdeI SnaBI
ligation E. coli XL1 colony poolP. putida KT2440
Po-Km/Po-sacB
Growthon plates
Conjugation
M9 succ Km + new effector
A domain Shuffling libraryNdeI/SnaBI
Sucrose
Plasmid extraction
P. putida KT2440 Po-luxAB Light emission
Genetic screening/selection
No Km
3-nt decanal test assayLB+3NT
LB+3NT
no inducer
no inducer
C-
C-
XylR 1
XylR 2
XylR 3
XylR 4
XylR 5
XylRwt
no inducer m-xylene phenol benzene
XylR 1
XylR 2
XylR 3
XylR 4
XylR 5
XylRwt
2-nt 3-nt 4-nt biphenyl
Visualization of effector-specificity changes
Sequence analysis of hybrid regulators
XylR1
XylR5
XylR DmpR
1 V124A 161-166 220
XylR2
F65L
XylR3
46-50
XylR4
L184I
161-166
In vivo activity of hybrid regulators m
-xile
nobe
ncen
o0
2
4
6
8
2 3 4 5 6 7 8 91XylR wt
no in
d-
feno
l
2-N
T3-
NT
4-N
Tbi
feni
lo 0
2
4
6
8
XylR12 3 4 5 6 7 8 91
no in
d-
feno
l
2-N
T3-
NT
4-N
Tbi
feni
lo 0
2
4
6
8
XylR22 3 4 5 6 7 8 91
no in
d-m
-xil
feno
lbe
nc2-
NT
3-N
T4-
NT
bife
nilo
0
2
4
6
8
XylR32 3 4 5 6 7 8 91
no in
d-m
-xil
feno
lbe
nc2-
NT
3-N
T4-
NT
bife
nilo
no in
d-m
-xil
feno
lbe
nc2-
NT
3-N
T4-
NT
bife
nilo0
2
4
6
8
XylR42 3 4 5 6 7 8 91
0
2
4
6
8
XylR52 3 4 5 6 7 8 91
no in
d-m
-xil
feno
lbe
nc2-
NT
3-N
T4-
NT
bife
nilo
m-x
ilbe
nc
m-x
ilbe
nc
Pro
mote
r act
ivit
yr
Pu
(b
-Gal, x
10
3)
(Apparent) affinity assays
0
2
4
6
8
110-4 10-3 10-2 10-1
XylR wt
XylR2XylR1
0
2
4
6
8
XylR2XylR1
3-MBA (mM) 3-NT (mM)
Pro
mote
r act
ivit
y P
u (
b-G
al, x
10
3)
XylR wt
110-4 10-3 10-2 10-1
Inhibition by 3-NT
0
1
2
3
4
5
6
0 0.5 1 1.5 2 2.5 3
3-NT (mM)
3-MBA 1 mM
XylR wt
Pro
mote
r act
ivit
y P
u (
b-G
al, x
10
3)
Structural prediction for the A domain of XylR
Grupo de Diseño de Proteínas-CNB
Contact surfaces protein/effector
Mapping structural changes in the model
XylR2
F65L
XylR3
46-50
XylR5
161-166
XylR2
F65L
XylR3
46-50
XylR5
161-166
Mapping structural changes in the model
XylR2
F65L
XylR3
46-50
XylR5
161-166
Mapping structural changes in the model
XylR1
XylR4
V124A
L184I
Mapping structural changes in the model
XylR1
XylR4
V124A
L184I
Mapping structural changes in the model
XylR wt
XylR5XylR4
XylR1
XylR3
XylR2
Loops involved in the effector pocket
Conclusions
• Mutants XylR1 to XylR5 bear changes that unlock the ability of XylR to respond to many non-natural effectors
• The changes involve not only the shape of the effector pocket, but also the structural transmission caused by inducer binding
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