Transcript Document 7376917

Project Overview
Landing Pads
Divide-by two Circuit
•Genetic toggle switch
switchable by one input
•Essential computing
component
Modeling
•General, useful tool for all synthetic biologists
•A systematic, Biobrick-compatible
approach to put plasmid constructs onto the
chromosome
Fabrication
•Characterize system •Construct gene operons in
•Determine robustness plasmid form and assemble
into the whole system
and predict ways to
improve performance
Insert DBT constructs
into landing pad and
cross into chromosome
Divide-By-Two Circuit
Idealized Divide-By-Two Circuit
Output (normalized)
input
Time
• The same input
toggles the system
between both
states
(on and off)
• Frequency of the
output
is half of the
frequency
of the input
• Output can be any
gene expression
DBT Circuit:
Single Input Toggle Switch
σ54
NR1
GFP
lacp
araB
gfp
lacl
glnG
cI
glnKp
rpoN
LacI
araBp
RFP
cl
nifHp
σ54
cl
cIp
nifA
nifA
lacl
rfp
How It Works
NR1
GFP
lacl
lacp
gfp
glnG
cI
lacl
rfp
glnKp
rpoN
araBp
nifHp
cl
cIp
nifA
How It Works
σ54
NR1
GFP
araB
lacl
lacp
gfp
glnG
cI
lacl
rfp
glnKp
rpoN
araBp
nifHp
σ54
cl
cIp
nifA
How It Works
σ54
NR1
GFP
lacp
lacl
gfp
glnG
cI
lacl
rfp
glnKp
rpoN
LacI
araBp
nifHp
σ54
cl
cIp
nifA
How It Works
σ54
lacp
lacl
gfp
glnG
cI
lacl
rfp
glnKp
rpoN
LacI
araBp
nifHp
σ54
cl
cIp
nifA
How It Works
lacp
lacl
gfp
glnG
cI
glnKp
rpoN
RFP
araBp
nifHp
cl
cIp
nifA
nifA
lacl
rfp
How It Works
σ54
araB
lacp
lacl
gfp
glnG
cI
glnKp
rpoN
RFP
araBp
nifHp
σ54
cl
cIp
nifA
nifA
lacl
rfp
How It Works
σ54
lacp
lacl
gfp
glnG
cI
glnKp
rpoN
RFP
araBp
cl
nifHp
σ54
cl
cIp
nifA
nifA
lacl
rfp
How It Works
σ54
lacp
lacl
gfp
glnG
cI
lacl
rfp
glnKp
rpoN
araBp
cl
nifHp
σ54
cl
cIp
nifA
How It Works
NR1
GFP
lacl
lacp
gfp
glnG
cI
lacl
rfp
glnKp
rpoN
araBp
nifHp
cl
cIp
nifA
Modeling
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Basic ODE’s
VglnKp ( 54 ) nsl ( NR1)
ngl
VcIp K cl ncl
d (lacI )

 ncl
  lacI (lacI )
ngl
nsl
n
n
cl
n
dt
( K sl  ( 54 ) sl )( K gl  ( NR1) gl ) K cl  cI
VNifHp ( 54 )nsc ( NifA)nnc
Vlacp Klc nlc
d (cI )

 nlc
  cI (cI )
nsc
nsc
nnc
nnc
nlc
dt
( Kbd  ( 54 ) )( K nc  ( NifA) ) Klc  lacI
•Hill function and degradation
•No Basal production
Steady State Analysis
• Under which conditions will the system
toggle and which conditions will it not?
• If it can toggle, how “easy” is it to
achieve this?
• How robust is the system?
Reducing Variable Dimensions
σ54
NR1
GFP
lacp
araB
gfp
lacl
glnG
cI
glnKp
rpoN
LacI
araBp
RFP
cl
nifHp
σ54
cl
cIp
nifA
nifA
lacl
rfp
Steady State Equations
lacI 
cI 
VcIp K cl
•Two variables and 8
parameters
ncl
ncl
 lacI ( K cl  cI )
ncl
Vlacp K lc
nlc
nlc
 cI ( K lc  lacI )
nlc
•Generally the parameters
are either unknown and/or
vary over a range
•Symmetric vs unsymmetric
Existence of Multiple Steady States
NO INPUT
3 steady
1 steady
states:
state:
2 stable,
stable 1 unstable
cI
Phase plane: one
threesteady
steadystate
states
lacI
Toggle System Needs Two Steady States
With INPUT
Two
stable
steady
toggle
Single
steady
state:states:
no toggle
canpossible
happen
Input added:
Inputtoggle
added:between
no toggle
two states
cI
GFP
RFP
lacI
What Parameter Ranges Yield
Toggle Activity?
• Only a narrow range of parameter values give
toggle behavior
• How robust is the system?
– Not very
• This will be addressed later
So how do you toggle?
Vary parameters
assuming…
No toggle
possible
Toggle
possible
•How does input affect the system?
•Is a toggle easily achieved?
System responds differently to varying input levels
Sufficient
Low input
Excess
input
input
Response of lacI and cI: insufficient
sufficient
excess input
input
input
concentration
Steady state 1
Transient
response
Ideal!
Quasi-steady state
QuasiSteady state 2
1
bifurcation
???
time
The Nature of Quasi-Steady State
Original system:
Two stable steady states
ONE quasiSteady state
Output 1
Back to original
steady states
Output 1
Output 2
High s54 level
Excess input
added
S54 degrades
Output 2
Quasi-Steady State Heavily
Favors One Output
End: Output 1
Start: Output
NOT
1 TOGGLE BEHAVIOR!!!
2
Output 1
Output 1
Output 2
High s54 level
Excess input
added
S54 degrades
Output 2
Dynamic Modeling of the DBT
• State transition takes
place
1. Rapidly increases
2. Dips back down
3. Slowly rises to
dominant level
Concentration (molecules)
• “Overthrowing” side:
lacI
cI
Time (s)
Dynamic Modeling of the DBT
Concentration
lacI
cI
Time
Potential Problem Spots
A: Input promoter must
be very quiet
A
B: Cross-talk between
the two values must be
low
C: lacp and cIp side
parameter values must
be relatively close
B
C
• Device predicted to
tend to have lacdominant side more
stable
• Can switch into lacdominant side, but
can’t switch out
Concentration (molecules)
• Large difference in
the parameters
between sides
Concentration (molecules)
Unbalanced, Gardner-based Simulations:
lacI: (blue)
cI:
(gold)
rfp:
(red)
gfp: (green)
Time (s)
lacI: (blue)
cI:
(gold)
rfp:
(red)
gfp: (green)
Time (s)
Stochastic Modeling
•Mass-Action Model and Gillespie Method
Tuning the Device with IPTG and Temperature
• Attenuate Lac with
IPTG
• Attenuate cI with heat
• Calibrate device by
varying both IPTG and
temperature
• Current research
focusing on if and
where “sweet-spot” is
located
Clamping the DBT
• Regardless of input pulse length/decay rate, only one
change-of-states occurs
Divide-By-Two Fabrication
Operons of the DBT Circuit
• Initial construction requires five plasmids, each carrying one operon of
the circuit
• Our final design will have fewer plasmids, as we will place some on the
chromosome and combine others
cIp-nifA-lacI-RFP
BBa_I720004
BBa_I720005
lacp-GFP-glnG-cI
lacp-GFP-glnG-cI (cont)
• Sequencing and characterization failed
– BioBrick had bad DNA (wrong lacp sequence)
– Actually needed repressible, rather than constitutive, promoter
– Sequencing results verified plasmid had all the genes, though
BBa_I720006
BBa_I720002
PCR: NCM 77
PCR: K. Pneumoniae genome
BBa_I720003
BBa_I720000
BBa_I720001
• Characterization:
– transformed plasmid into rpoN mutants: growth regardless of arabinose
input
– Also transformed rpoN alone: still have growth
– Too noisy; need a single copy
– Back-up plan: tetO-rpoN
DBT Operons in Landing Pads
Synthetic Operon
Landing Pad Used
Leucine BioBrick
Landing Pad
BBa_I720007
Arabinose BioBrick
Landing Pad
glnK Landing Pad
DBT Operons in Landing Pads
BioBrick Landing Pad
Built so that
ANYONE can insert
ANY BioBrick onto the
Chromosome of E.
Coli!
BioBrick Landing Pad:
Goals of Project
• General Landing Pad Goal: Aid in insertion of constructs onto
the chromosome
• Develop a general method for constructing landing pads that:
• Have BioBrick compatible restriction sites
• Allow easy phenotypic screening
• Limit noise
• Allow nesting of sequential landing pads
BioBrick Landing Pad
BioBrick Landing Pad
BioBrick Landing Pad:
Homologous Regions
Arabinose
Homologous Regions
Homologous
Recombination of
Arabinose Landing Pad
BioBrick Landing Pad: Nesting
Landing Pads
Benefits of Nested Landing Pads
• Allow for more constructs to be inserted onto the
chromosome at the same chromosomal location
• Different drug resistance gene is used: Only one type
of screening is required
BioBrick Landing Pad: Nesting
Landing Pads
New Landing Pad
Criteria
• Homologous regions
from previous drug
resistance gene (ChlorR)
• Different drug
resistance gene (KanR)
BioBrick Landing Pad:
Nested Landing Pads
Nested Product: Chloramphenicol Landing Pad
nested in Arabinose Landing Pad
BioBrick Landing Pad:
Fabrication Progress
Landing Pads
250 bp Arabinose Landing Pad
500 bp Arabinose Landing Pad
1000 bp Arabinose Landing Pad
250 bp Maltose Landing Pad
500 bp Maltose Landing Pad
1000 bp Maltose Landing Pad
Status
Completed
Completed
Completed
In Progress
In Progress
In Progress
Registry Number
BBa_I720008
BBa_I720009
BBa_I7200010
N/A
N/A
N/A
* Nesting to be tried in the near future as well!
• Prof. Alex Ninfa
• Domitilla DelVecchio
• Prof. Peter Woolf
• Dong Eun Chang
Questions?
Unbalanced System: Bifurcation
favors one state
• In a more
realistic
unbalanced
system, the
quasi-SS leads
to one of the
two steady
states
• One steady
state is
favored over
the other
Quasi-Steady State Bifurcates
with Sigma54 level (Balanced System)
• If the system
is balanced,
as sigma54
degrades past
threshold both
steady states
are equally
accessible
Stable SS
Unstable SS
Quasi SS
Sigma54 concentration
Low input: no sigma54 presence after response
Time