TriggeringPigment Produc/onin E.Coli - - PowerPoint PPT Presentation







Triggering
Pigment
 Produc/on
in
E.
Coli


Cambridge
2009


Mike
Davies,
Shuna
Gould,
Siming
Ma,
Vivian
Mullin,
 Megan
Stanley,
Alan
Walbridge,
Crispian
Wilson
 Celebra2ng
800
Years
of
Innova2on
at
Cambridge
University


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




Lack
of
self‐contained
output


– Reliance
on
reporters
in
 Registry
 – Require
addi/onal

 technology
to
read
output


Colour
Generators 


– Bacterial
pigments
 – Visible,
user‐friendly
output


Bacterial
Biosensors:
Problems



Inability
to
tune
sensor
as
 desired


– Limited
by
sensi/vity
of
 promoter
 – Limited
to
PoPS
output
 behaviour
of
promoter


S
 E
 N
 S
 O
 R


R
 E
 P
 O
 R
 T
 E
 R


S
 E
 N
 S
 O
 R


R
 E
 P
 O
 R
 T
 E
 R


T
 U
 N
 E
 R
 C
 O
 L
 O
 U
 R


Bacterial
Biosensors:
Solu2ons



SensiHvity
Tuners


– PoPS
converters
 – Change
sensi/vity
of
 upstream
promoter


T
 U
 N
 E
 R
 C
 O
 L
 O
 U
 R
 Cambridge
2009


Bacterial
Biosensors:
Easy
to
use


S
 E
 N
 S
 O
 R
 T
 U
 N
 E
 R
 C
 O
 L
 O
 U
 R
 Cambridge
2009










































Inducer
concentra/on:
 






















0






























low




























high
 



The
colour
readout
indicates
concentra/on
of
inducer


Bacterial
Biosensors:
A
prototype


Cambridge
2009


PoPS


SENSOR Input  PoPS Receiver SENSITIVITY TUNER PoPS  PoPS Converter COLOUR GENERATOR PoPS  Colour Reporter

Input
 PoPS


Bacterial
Biosensors:
How
to
build
a


bacterial
biosensor
with
these
parts


Cambridge
2009
 COLOUR
 GENERATOR
 SENSITIVITY
TUNER
 Ac/vator
 sensi/ve
 promoter
 Phage
 ac/vator
 SENSOR
 Chemical
 IN
 Pigment
 OUT
 Pigment
 producing
 device

 Promoter
 sensi/ve
to
 input


T

• A
Sensi/vity
Tuner


allows
adjustment
of
 sensi/vity
to
input


• A
combina/on
of


different
Tuners
in
 parallel
allow
 measurements
of
a
 range
of
discrete
input
 concentra/ons


Concentra/on
 Rate
of
Output


SensiHvity
Tuners:
Introduc2on


Cambridge
2009


PoPS
in
 Transcrip/onal
and
 Transla/onal
 Characteris/cs
 Promoter
 Characteris/cs

 Ac/vator
 Concentra/on
 PoPS
out


Design:
an
Input
to
Output
Device


Cambridge
2009
 Ac/vator
 sensi/ve
 promoter
 Phage
 ac/vator


T

PoPS
in
 PoPS
out


“Amplifiers”


• GFP
output
controlled
by
phage


promoter


• RFP
output
controlled
by
pBad


input


• Characterized
as
an
“amplifier”
by


ra/o
of
RFP
to
GFP


ac/vators
 promoters


P2
ogr
 PSP3
pag
 phiR73
delta
 PF
promoter

 I746370
 I746380
 I746390
 PO
promoter
 I746371
 I746381
 I746391
 PP
promoter
 I746372
 I746382
 I746392
 Psid
promoter
 I746374
 I746384
 I746394
 PLL
promoter
 I746375
 I746385
 I746395


Previous
Work:
Cambridge
2007


Cambridge
2009
 Ac/vator
 sensi/ve
 promoter
 Phage
 ac/vator


T

I13507
 mRFP
 I13504
 GFP
 I0500
 pBad/AraC


PoPS
in
 Phage
Ac/vator
 Transcrip/on
&
 Transla/on
 Characteris/cs
 Phage
 Promoter
 Characteris/cs

 Ac/vator
 Conc.
 PoPS
out
 pBAD
 Promoter
 Characteris/cs

 GFP
 Transcrip/on
 &
Transla/on
 Characteris/cs
 Arabinose
 Conc.
 GFP
 Conc.
 where


• Model
gene
characteris/cs
at
steady


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


SensiHvity
Tuners:
Modelling


Cambridge
2009


pBAD
 Promoter
 Characteris/cs

 Phage
 Promoter
 Characteris/cs

 Phage
Ac/vator
 Transcrip/on
&
 Transla/on
 Characteris/cs
 GFP
 Transcrip/on
 &
Transla/on
 Characteris/cs


• 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


Modelling
Results:
Sigmoidal
Behaviour


Cambridge
2009


Reporter Degradation rates at multiple input concentrations of arabinose

time Reporter production rate

Model for maximum fluorescence rate

Inducer concentration Reporter production rate

Rate
of
GFP
expression
 Concentra/on
of
Arabinose
 Increase
in
rate
(a)
 Half‐maximal
induc/on
(k)
 




Hill

 coefficient
 





(n)
 1
RPU
 Peak
rate
 Basal
rate
(c)



Curve
FiTng:
Hill
Func2on


Cambridge
2009
 A model Sensitivity Tuner

pBAD -> GFP

Arabinose concentraion (µm) Maximum normalised GFP production

• Constructs
were
tested
on
high
copy
against
pBAD


characteris/cs


• Output
triggered
at
much
lower
arabinose


concentra/on
when
Sensi/vity
Tuner
included


SensiHvity
Tuners:
Changing
the


sensi2vity
of
an
upstream
promoter


Cambridge
2009
 pBAD -> Construct 91 -> GFP

Arabinose concentraion (µm) Maximum normalised GFP production

P2
ogr
 PSP3
pag
 phiR73
delta
 PF
promoter

 I746370
 I746380
 I746390
 PO
promoter
 I746371
 I746381
 I746391
 PP
promoter
 I746372
 I746382
 I746392
 Psid
promoter
 I746374
 I746384
 I746394
 PLL
promoter
 I746375
 I746385
 I746395


• 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


measured


• Standard
Promoter
included

• n
plate
to
allow
for
RPU


measurements


SensiHvity
Tuners:
Characterisa2on


Cambridge
2009


• Matlab
graphical
interface
developed
to
allow
data
to
be


viewed
in
several
ways


• Standard
promoter
data
allows
for
RPU
characterisa/on


SensiHvity
Tuners:
SoLware


Cambridge
2009


• 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


Curve
FiTng:
Hill
Func2on


Cambridge
2009


• A
range
of
Sensi/vity
Tuners
were
successfully
characterised

• n
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


Concentra/on
of
Arabinose
 Rate
of
GFP
expression
 Increase
in
rate
(a)
 Half‐maximal
induc/on
(k)
 




Hill

 coefficient
 





(n)
 1
RPU
 Peak
rate
 Basal
rate
(c)



SensiHvity
Tuners:
Parameters


Cambridge
2009
 A model Sensitivity Tuner

• 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
 PP
promoter
 K274382
 K274392
 Psid
promoter
 K274374
 K274384
 K274394
 PLL
promoter
 K274375
 K274395


SensiHvity
Tuners:
Design


Cambridge
2009


promoter
 ac/vator


T

• Diversity:


– Colour
 – Bacterial
Origin


• Design


– Standard
Assembly
 – PCR
 – Synthesis


• PotenHal
for
ManipulaHon


– Single
gene
systems
 – Mul/gene
systems
with
 colourful
intermediates
 – Supplements
to
media


Violacein
 Melanin
 Carotenoids


Colour
Generators:
Choosing
pigments


Cambridge
2009


L-tryptophan

• Quorum‐sensing


controlled
pigment
from
 Chromobacterium
 violaceum


Violet
 Green


VioD VioA VioB VioE VioC

Violacein:
Background


Cambridge
2009


Violacein:
Design
&
Synthesis


VCG
 K274002
 K274002
 BamHI BglII BclI

VioD VioA VioB VioC VioE

Cambridge
2009


Violacein:
Design
&
Synthesis


VCG
 K274002
 K274002
 BamHI BglII BclI

VioA VioB VioC VioD VioE

A G A T C T T C T A G A G G A T C C C C T A G G A G A T C T T C T A G A G G A T C C C C T A G G Cambridge
2009


K274003


Violacein:
Design
&
Synthesis


VCG
 K274002
 K274002
 BamHI BglII BclI

VioA VioB VioC VioD VioE VioA VioB VioC VioD VioE VioA VioB VioC VioD VioE

GCG
 K2742003
 G A T C T A G C C T A G Cambridge
2009


Violacein:
Expression
&
Quan2fica2on


VCG
 K274002
 GCG
 K2742003
 Cambridge
2009


584


Violacein:
Expression
&
Quan2fica2on


Cambridge
2009


A VioA, VioB, VioD, VioE VioC B Colour Output A
 B
 Output
 0
 0
 No
colour
 1
 0
 GREEN
 0
 1
 No
colour
 1
 1
 VIOLET


Violacein:
Colour
Logic


If A = constitutive, B = inducible Colour Output Device working: Presence of B: Cambridge
2009


• Brown
pigment
made
in
many
animals


 and
bacteria
via
the
ac/on
of
a
 tyrosinase
(MelA)


• Isolated
melA
from
Rhizobium
etli.

• Media
supplemented
with
copper


sulphate
and
tyrosine


Melanin:
Background


Brown


MELANIN


Tyrosine
 Dopaquinone


MelA

polymerisation Cambridge
2009


• Used
na/ve
RBS
and
planned
to


remove
forbidden
restric/on
sites
 using
PCR


• Strong
candidate
for
a
biosensor


reporter
 − Strong
pigment
produc/on
 − Single
gene


Melanin:
Design


BCG
 K274001
 K274001
 Native rbs MelA Cambridge
2009


Lycopene β-Carotene

Carotenoids:
Background


Cambridge
2009


Lycopene β-Carotene

Carotenoids:
Background


Cambridge
2009


pyruvate glyceraldehyde-3-phosphate Farnesyl pyrophosphate (Colourless precusor) Non-mevalonate Pathway (already present in E. coli)

CrtE CrtB CrtI CrtY

Lycopene β-Carotene

Carotenoids:
Background


Cambridge
2009


Enzymes coding sequences from Pantoea ananatis (Enterobacteria)

Farnesyl pyrophosphate (Colourless precusor)

CrtE CrtB CrtI CrtY

Lycopene β-Carotene

RCG
 K274100
 OCG
 K274200


Carotenoids:
Standard
assembly


Cambridge
2009


RCG
 K274100
 OCG
 K274200
 RCT
 K274110
 OCT
 K274210


Constitutive promoter

Expression in E. coli strain MG1655

Carotenoids:
Standard
assembly


Cambridge
2009


RCT
 K274110
 OCT
 K274210


Carotenoids:
Expression
and
Quan2fica2on


Cambridge
2009


β-Carotene: 1.5 µg per mL culture

Lycopene Control β-Carotene Control 5µg carotene

474 456

Carotenoids:
Expression
and
Quan2fica2on


Cambridge
2009


SENSOR
 Promoter
 sensi/ve
to
 input
 COLOUR
 GENERATOR
 Pigment
 producing
 device

 Ac/vator
 sensi/ve
 promoter
 Phage
 ac/vator


T

SENSITIVITY TUNER

I0500 CrtE CrtB CrtI I0500 CrtE CrtB CrtI CrtY

Proof
of
Concept:
Pigment
Induc2on


Pbad promoter

IRCT
 K274120
 IOCT
 K274220


Cambridge
2009


1mM arabinose No arabinose

β-Carotene: 1.3 µg per mL induced culture

No arabinose Induced by 1mM arbinose Control 5 µg carotene

456

Proof
of
Concept:
Pigment
Induc2on


Cambridge
2009


P2 ogr PSP3 pag phiR73 delta PF promoter PO promoter PP promoter Psid promoter PLL promoter

✗

✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔

K274370 K274371 K274374 K274375 K274380 K274381 K274384 K274382 K274391 K274392 K274395 K274394

✗ ✗

BioBricks:
Sensi2vity
Tuners


ac/vator

 





promoter
 ST

Cambridge
2009


BioBricks:
Colour
Generators


BCG
 K274001
 RCG
 K274100
 OCG
 K274200
 VCG
 K274002
 GCG
 K2742003
 Cambridge
2009


BioBricks:
Systems


R0011 CrtE CrtB CrtI I0500 CrtE CrtB CrtI R0011 CrtE CrtB CrtI CrtY

RCT
 K274110
 OCT
 K274210
 IRCT
 K274120


I0500 CrtE CrtB CrtI CrtY

IOCT
 K274220


Cambridge
2009


• Show
compa/bility
with


promoters
in
Registry


• Expand
kit
of
parts


– Phage
ac/vators
and
phage
 promoters
 – Pigment‐producing
operons
 from
other
bacterial
species


Further
Work:
for
our
Project


Cambridge
2009


Arsenic Mercury Lead As + Hg Ag + Pb Hg + Pb As + Hg + Pb

MulHplexing
InformaHon:
Accessible


and
Informa2ve
Biosensors


Cambridge
2009


…would
like
to
say
a
few
thank
yous 
 The
Cambridge
2009
iGEM
team…


Cambridge
2009


…to
Jeremy
Minshull
and
his
colleagues
at
 DNA2.0
for
their
generous
offer
to
help
us
 build
and
synthesize
the
violacein
operon.


Thank
You…


Cambridge
2009


to
all
our
sponsors


Thank
You…


Cambridge
2009


Advisors:
 Dr.
Jim
Ajioka
 Dr.
Jim
Haseloff
 Dr.
Gos
Micklem
 Dr.
Tom
Ellis
 Dr.
Duncan
Rowe
 …and
especially
 James
Brown
 Friends:
 Caitlin
Cockerton
 Daisy
Ginsberg
 James
King
 Tuur
Van
Balen
 Summary of Achievements: Designed 23 New Biobricks Characterised 15 Biobricks already in the registry

Thank
You…


Cambridge
2009


Advisors:
 Dr.
Jim
Ajioka
 Dr.
Jim
Haseloff
 Dr.
Gos
Micklem
 Dr.
Tom
Ellis
 Dr.
Duncan
Rowe
 …and
especially
 James
Brown
 Friends:
 Caitlin
Cockerton
 Daisy
Ginsberg
 James
King
 Tuur
Van
Balen
 Summary of Achievements: Designed 23 New Biobricks Characterised 15 Biobricks already in the registry

Thank
You…


Cambridge
2009


It’s
Mike’s
birthday
today…hopefully
he’s
not
 looking
at
the
screen!
 We’d
like
to
sing
him
happy
birthday,
so
join
us!
 On
3…..


1 2 3!!!