Andrew Yang Jonathan Babb Ron Weiss 9232011 Null versus 1 Systems Circuits 16 NOTE Not depicted is a stretch of DNA following the promoters and preceding the genes called the RBS The RBS is tunable thus controlling the strength of gene expression Please account for this possibi ID: 374618
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Slide1
MURI Pattern Detection Circuits
Andrew Yang, Jonathan Babb, Ron Weiss
9/23/2011Slide2
Null versus 1 Systems (Circuits #1-6)
NOTE: Not depicted is a stretch of DNA following the promoters and preceding the genes called the ‘RBS’. The RBS is tunable, thus controlling the strength of gene expression. Please account for this possibility.
Null
Null
“1”
Circuit operation
: Initial state of
toggle set
for CI
production
with IPTG. Later, UV induces state change for a region by cleaving CI. If the region is large enough
to achieve an engineering quorum, the quorum is amplified to the entire region and reported by a blue fluorescent protein.
Goal: Digital behavior. Circle sizes will
be smaller or larger than aforbidden band.
UV Toggle 3OC12HSL Quorum Sensor
3OC12HSL Propagator State “1” = 30C12HSLSlide3
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
LasI
LasR
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
Las System
Circuit #1Slide4
Circuit #2: External atc
->
LasR
atc
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
LasI
LasR
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
ptet
TetR
pLacIqSlide5
Circuit #3: atc
->
LasI
Activator
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pActivator
LasI
LasR
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
ptet
TetR
pLacIq
pLas
Activator
atcSlide6
Circuit #4:
Const.
Lactonase
atc
->LasR
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
LasI
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
LasR
ptet
TetR
pLacIq
AiiA
pConstitutive
atcSlide7
Circuit #4A: Const.
Lactonase
No
atc
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
LasI
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
LasR
pLacIq
AiiASlide8
Circuit #5:
atc
->
Lactonase
Positive Feedback LasI
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
Las
R
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
Las
I
ptet
TetR
pLacIq
AiiA
atcSlide9
Circuit #6:
atc
->Inducible
Lactonase
Positive Feedback LasI
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
Las
R
G
FP
30C12HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
Las
I
pLas
/
tetO
TetR
pLacIq
AiiA
atcSlide10
Null
Null
“1”
“2”
Null versus 1 versus 2 Systems (Circuit #7)
NOTE: As before, not depicted is a stretch of DNA following the promoters and preceding the genes called the ‘RBS’. The RBS is tunable, thus controlling the strength of gene expression. Please account for this possibility.
Key:
LasR
has low sensitivity and only detects large circles (2) while
RhiR
system has high sensitivity for small circles (1).
TetR
represses small circles if when large are also present.
Operations: now we have two separate quorum sensing systems tuned to different sensitivities. To detect large circles,
LasR
will amplify with a low sensitivity. In the case where there is no large circle, a more sensitive
RhiR system will detect and amplify a smaller circle. Forbidden bands in between each range are not unused by the digital input.
UV Toggle 3OC12HSL Quorum Sensor
3OC12HSL Propagator C4HSL Quorum Sensor
C4HSL Propagator State “1” = C4 ^ C12 State “2” = C12 Null = NOR(C4,C12)Slide11
Circuit #7: Null versus 1 versus 2 system
CI
pLasORI
LasI
LasR
mCherry
LacI
pLac
pCI
UV
30C12HSL
IPTG
pLas
LasI
LasR
B
FP
30C12HSL
Quorum Sensors
Propagator
Toggle Switch
pRhiORI
RhiI
RhiR
mCherry
C4HSL
pRhi
RhiI
RhiR
G
FP
C4HSLSlide12
Appendix
Following are circuit #1 with the
Rhil
and Lux systems. The same changes in promoters, inducers, and repressors can be applied to circuits #2-6.Slide13
Rhil
System
CI
pRhiORI
RhiI
RhiR
mCherry
LacI
pLac
pCI
UV
C4HSL
IPTG
pRhi
RhiI
RhiR
G
FP
C4HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
Circuit #1Slide14
CI
pLuxORI
LuxI
LuxR
mCherry
LacI
pLac
pCI
UV
C6HSL
IPTG
pLux
LuxI
LuxR
G
FP
C6HSL
Quorum Sensor
Propagator
Toggle Switch
Quorum sensor activated only in UV exposed region.
Lux System
Circuit #1