TriggeringPigment Produc/onin E.Coli - PowerPoint PPT Presentation

Cambridge
2009
 




 
Triggering
Pigment
 Produc/on
in
 E.
Coli
 Mike
Davies,
Shuna
Gould,
Siming
Ma,
Vivian
Mullin,
 Megan
Stanley,
Alan
Walbridge,
Crispian
Wilson
 Celebra2ng
800
Years
of
Innova2on
at
Cambridge
University


Cambridge
2009
 The
Cambridge
2009
iGEM
team
has
created
a
 Kit
of
Parts
 that
will
facilitate
the
 design
 and
 construc1on
 of
 biosensors
 in
the
 future 
 We
have
developed
a
set
of
 Sensi1vity
Tuners
 and
a
set
of
 Colour
Generators 


Cambridge
2009
 Bacterial
Biosensors:
 the
Detec2on
of
 Environmental
Pollutants
 • Bacterial
biosensors
‐
an
 alterna/ve
to
chemical
 methods
 • S/ll
selec/ve
and
 sensi/ve
 • Inexpensive
 • Less
labour
intensive
 • More
accessible


Cambridge
2009
 Bacterial
Biosensors:
 Problems
 Bacterial
Biosensors:
 Solu2ons
 R
 R
 S
 S
 C
 C
 T
 T
 E
 E
 E
 O
 E
 O
 P
 P
 U
 U
 N
 N
 L
 L
 O
 O
 N
 N
 R
 S
 S
 R
 O
 O
 E
 E
 T
 T
 O
 O
 U
 U
 R
 R
 E
 E
 R
 R
 R
 R
 R
 R
 

Lack
of
self‐contained
output
 
Inability
to
tune
sensor
as
 
 SensiHvity
Tuners
 Colour
Generators 
 desired 
 – Limited
by
sensi/vity
of
 – PoPS
converters
 – Reliance
on
reporters
in
 – Bacterial
pigments
 promoter
 – Change
sensi/vity
of
 Registry
 – Visible,
user‐friendly
output
 – Limited
to
PoPS
output
 upstream
promoter
 – Require
addi/onal

 behaviour
of
promoter
 technology
to
read
output


Cambridge
2009
 Bacterial
Biosensors:
 Easy
to
use
 S
 C
 T
 E
 O
 U
 N
 L
 N
 S
 O
 E
 O
 U
 R
 R
 R


Cambridge
2009
 Bacterial
Biosensors:
 A
prototype
 







































Inducer
concentra/on:
 






















0






























low




























high
 



The
colour
readout
indicates
concentra/on
of
inducer


Cambridge
2009
 Bacterial
Biosensors:
 How
to
build
a
 bacterial
biosensor
with
these
parts
 Pigment
 Chemical
 IN
 OUT
 COLOUR
 SENSOR
 SENSITIVITY
TUNER
 GENERATOR
 Input
 SENSOR T PoPS
 SENSITIVITY PoPS
 TUNER Phage
 Input  PoPS COLOUR Ac/vator
 Promoter
 Pigment
 Receiver ac/vator
 GENERATOR PoPS  PoPS sensi/ve
to
 sensi/ve
 producing
 Converter promoter
 input
 device

 PoPS  Colour Reporter

Cambridge
2009
 SensiHvity
Tuners:
 Introduc2on
 Rate
of
Output
 • A
Sensi/vity
Tuner
 allows
adjustment
of
 sensi/vity
to
input
 Concentra/on
 • A
combina/on
of
 different
Tuners
in
 parallel
allow
 measurements
of
a
 range
of
discrete
input
 concentra/ons


Cambridge
2009
 Design:
 an
Input
to
Output
Device
 PoPS
in
 PoPS
out
 T Phage
 Ac/vator
 ac/vator
 sensi/ve
 promoter
 Ac/vator
 PoPS
in
 Transcrip/onal
and
 Concentra/on
 PoPS
out
 Promoter
 Transla/onal
 Characteris/cs

 Characteris/cs


Cambridge
2009
 Previous
Work:
 Cambridge
2007
 T I0500
 Phage
 Ac/vator
 I13504
 I13507
 pBad/AraC
 ac/vator
 sensi/ve
 GFP
 mRFP
 promoter
 “Amplifiers”
 ac/vators
 • GFP
output
controlled
by
phage
 P2
ogr
 PSP3
pag
 phiR73
delta
 promoter
 PF
promoter

 I746370
 I746380
 I746390
 promoters
 PO
promoter
 I746371
 I746381
 I746391
 • RFP
output
controlled
by
pBad
 PP
promoter
 I746372
 I746382
 I746392
 input
 Psid
promoter
 I746374
 I746384
 I746394
 PLL
promoter
 I746375
 I746385
 I746395
 • Characterized
as
an
“amplifier”
by
 ra/o
of
RFP
to
GFP


Cambridge
2009
 SensiHvity
Tuners:
 Modelling
 Arabinose
 Ac/vator
 GFP
 PoPS
in
 Phage
Ac/vator
 Phage
Ac/vator
 GFP
 GFP
 pBAD
 pBAD
 Phage
 Phage
 Conc.
 Conc.
 PoPS
out
 Conc.
 Transcrip/on
&
 Transcrip/on
&
 Transcrip/on
 Transcrip/on
 Promoter
 Promoter
 Promoter
 Promoter
 Transla/on
 Transla/on
 &
Transla/on
 &
Transla/on
 Characteris/cs

 Characteris/cs

 Characteris/cs

 Characteris/cs

 Characteris/cs
 Characteris/cs
 Characteris/cs
 Characteris/cs
 Model
gene
characteris/cs
at
steady
 • where
 state
using
Law
of
Mass
Ac/on
 pBAD
is
repressed
by
repressor
X*
 • which
binds
to
arabinose
 • Assume
transcrip/on
and
transla/on
 are
linear
func/ons
of
PoPS
 Model
protein
concentra/ons
as
 • dynamic,
since
these
change
slowly
 Allow
for
protein
degrada/on
 •

Cambridge
2009
 Modelling
Results:
 Sigmoidal
Behaviour
 • The
model
contains
a
large
number
of
constants
 • A
priori 
modelling
requires
arbitrary
values
to
be
chosen
 • Maximum
reporter
produc/on
rate
is
sigmoidal
with
inducer
 concentra/on
 Reporter Degradation rates at multiple input Model for maximum fluorescence rate concentrations of arabinose Reporter production rate Reporter production rate Inducer concentration time

Cambridge
2009
 Curve
FiTng:
 Hill
Func2on
 A model Sensitivity Tuner Peak
rate
 1
RPU
 Rate
of
GFP
expression
 




Hill

 coefficient
 Increase
in
rate
(a)
 





(n)
 Basal
rate
(c)

 Half‐maximal
induc/on
(k)
 Concentra/on
of
Arabinose


SensiHvity
Tuners:
 Changing
the
 Cambridge
2009
 sensi2vity
of
an
upstream
promoter
 • Constructs
were
tested
on
high
copy
against
pBAD
 characteris/cs
 • Output
triggered
at
much
lower
arabinose
 concentra/on
when
Sensi/vity
Tuner
included
 pBAD -> GFP pBAD -> Construct 91 -> GFP Maximum normalised GFP Maximum normalised GFP production production Arabinose concentraion ( µ m) Arabinose concentraion ( µ m)

Cambridge
2009
 SensiHvity
Tuners:
 Characterisa2on
 • 15

Cambridge
2007
 constructs
moved
down
to
 low
copy
plasmid
 • High
throughput
tes/ng
 • 3
repeats
of
3
colonies
over
 8
concentra/ons
 • OD
and
fluorescence
 P2
ogr
 PSP3
pag
 phiR73
delta
 measured
 PF
promoter

 I746370
 I746380
 I746390
 PO
promoter
 I746371
 I746381
 I746391
 • Standard
Promoter
included
 PP
promoter
 I746372
 I746382
 I746392
 on
plate
to
allow
for
RPU
 Psid
promoter
 I746374
 I746384
 I746394
 PLL
promoter
 I746375
 I746385
 I746395
 measurements


Cambridge
2009
 SensiHvity
Tuners:
 SoLware
 • Matlab
graphical
interface
developed
to
allow
data
to
be
 viewed
in
several
ways
 • Standard
promoter
data
allows
for
RPU
characterisa/on


Cambridge
2009
 Curve
FiTng:
 Hill
Func2on
 • Non‐linear
least
squares
method
used
to
fit
Hill
 func/ons
to
measured
data
 • Each
fit
produces
the
parameters
of
the
Hill
func/on,
 enabling
construct
to
be
quan/ta/vely
analysed


Cambridge
2009
 SensiHvity
Tuners:
 Parameters
 • A
range
of
Sensi/vity
Tuners
were
successfully
characterised
 on
low
copy
 • Good
range
in
sensi/vity:
10x
range
in
half‐maximal
induc/on
 • Hill
coefficients
of
2
–
3
when
concentra/on
resolu/on
is
 sufficient
 • Wide
range
of
rate
increases,
from
0.3RPU
to
1.2RPU
 A model Sensitivity Tuner Peak
rate
 1
RPU
 




Hill

 Rate
of
GFP
expression
 coefficient
 Increase
in
rate
(a)
 





(n)
 Basal
rate
(c)

 Half‐maximal
induc/on
(k)
 Concentra/on
of
Arabinose


Cambridge
2009
 SensiHvity
Tuners:
 Design
 • A
standard
kit
was
designed
using
well
characterised
 candidates
 • Tuners
can
be
used
with
any
promoter
 • Any
device
can
be
placed
downstream
of
the
 construct
 P2
ogr
 PSP3
pag
 phiR73
delta
 PF
promoter

 K274370
 K274380
 PO
promoter
 K274371
 K274381
 K274391
 T PP
promoter
 K274382
 K274392
 ac/vator
 promoter
 Psid
promoter
 K274374
 K274384
 K274394
 PLL
promoter
 K274375
 K274395