Bacteria-to-Yeast Optical Communication: Using Light as a - - PowerPoint PPT Presentation

Bacteria-to-Yeast Optical Communication:

Using Light as a trans-Activating Factor to Bridge a Physically Split lac Operon

Optical Communication

Luciferase Enzyme + Luciferin + O2  Oxyluciferin + Light Light

Bacteria to Yeast Communication

Bacteria (Signal Sender) Light (Means of Communication) Yeast (Signal Receiver)

Splitting the lac Operon

Spatial Splitting of the Lac Operon

Der erepr epression ession T rans-activ ans-activating ating factor actor Beta-Gal Beta-Gal pr production

• duction

De-repression of the Operon

The Signal: Bioluminescence from Luciferase

Luciferase Enzyme + Luciferin + O2  Oxyluciferin + Light Light

Signal Receiver: Yeast

Signal Receiver: Yeast

Signal Receiver: Yeast

Spatial Splitting of the Lac Operon

Characterization of the Yeast-Two Hybrid System

LASERPETTOR

• To
 expedite
 the
 laser
 based
 characteriza2on
 experiments,
 a
 “laser‐pe8or”
 was


designed,
built
and
tested


• Simultaneously
irradiate
up
to
eight
samples
contained
within
a
96‐well
micro2ter


plate.

 Design


• Briefly,
when
the
switch
is
closed
the
circuit
ac2vates
the
~650nm

• AIer
2me
delay
diodes
switch
off

• Green
LED
turns
on
w/
beeper


• Control
2me
laser
diodes
remain
on
w/a
variable
resistor
and/or
switching


between
one
of
three
capacitors.

 Laser
Pe8or
 Laser
Diode
 Array
 Circuit
Diagram


PCB Extraction and Testing

• Phycocyanobilin

(PCB)
is
necessary
for
PhyB
func2onality

• Not
naturally
present
in
Yeast
Spirulina
Extrac2on

• Time‐course
Experiment
(see
if
PCB
is
toxic)


X-GAL Assays


• PhyB/PIF3
2
hybrid
system



– In
presence
of
red
fluorescent
light
and
PCB
Beta‐
galactosidase
is
 produced


• Performed
filter
liI
assay
using
nitrocellulose
filters
to
screen
for
Y190


colonies
with
2
hybrid‐system




• Successful
colonies
re‐streaked
onto
leu‐/trp‐


– Re‐screened
aIer
overnight
exposure
to
red
light
in
presence
of
PCB


Characterization: Laser Based Testing of System Sensitivity Biodot
Assay


• Immobilized
cells
from
liquid


culture
lysed
w/
liquid
nitrogen
 and
incubated
(30
C)overnight

 in
150uL
X
gal
buffer


• Also
looking
for
B‐Galactosidase


ac2vity


PCB
Concentra2on

 Assay


• Y190
cells
with
PhyB
DBD/PIF3


AD
grown
to
10^6
cells/mL


• plated
in
100uL
solid
media


• PCB
effec2ve
in
inducing
Lac
Z


expression


Characterization: Laser Based Testing

• Same
density
as
PCB


concentra2on
assay


• 650
nm
light
pulse

• Pulsed
cells
for
10
sec


and
incubated
for
30


• r
60
min

• 60
min

higher
B‐Gal


expression


Characterization: Cell Age

• Found
from
previous
laser
based
tes2ng

Assays
are


dependent
on
Cell
Age


• Older
cells
have
higher
background
in
nega2ve


control


• Y190
contains
B‐Gal
and
His
reporters
under
the


control
of
the
Gal
promoter



• Muta2ons
up‐regula2ng
His
produc2on



cons2tu2ve
beta‐gal
expression.


• Add
addi2onal
His


Characterization: Cell Concentration

• Cell
concentra2on
was
too
high

posi2ve
background.


• High
levels
of
beta
gal
(absence
of
induc2on)
is
greater
w/
higher
cell


concentra2on


• Dilu2on
of
a
culture
reduces
posi2ve
background.


• Assay

• Expose

cells
to
light
or
dark



• Sequen2al
dilu2ons
to
examine
the
effects
of
cell
concentra2on.


• Highest
concentra2on

background
on
nega2ve
control

• 10
fold
dilu2on
reduced
background

• Hard
to
see
color
change
with
further
dilu2ons


Bacteria to Yeast Communication: Variables To Contend With

Poten2al
Experimental
Result Variable
Responsible




+
Control
 


‐

Control
 


Experiment
 Poor
batch
quality
of
crude
PCB
 Extract
 


+
Control
 


‐

Control
 


Experiment
 Low
expression
levels
of
red
 luciferase
in
bacteria
(light
 emission
levels)
 


+
Control
 


‐

Control
 



Experiment
 Elevated
B‐gal
expression
level


• f
yeast
cultures
(problems
with


induced
background
 expression).


Successful Bacteria to Yeast Communication??

Experimental:

 Bacterial
Light
+
PCB
 Nega=ve
Control:
 Bacterial
Light
+
DMSO


Future directions

PCB Extraction

PCB Biosynthesis

Cellular Blackboard

• Red
Light
PhyB
DBD
+
PIF3AD

Luciferase
Expression

• Use
light
to
write
on
a
bacterial
lawn

• Far
Red
Light
PhyB
DBD

PIF3AD

No
expression

• Use
far
red
light
to
act
as
an
“eraser”


The Yeast Red-Light District

• Yeast
come
in
two
ma2ng
types:
matA
and
matAlpha

• mate
with
cells
of
opposite
ma2ng
types


• Switch
ma2ng
types
upon
ma2ng
(HO
Endonuclease)

• Lab
strains
are
HO
Endonuclease
knockouts,


• Red
light
ma2ng
type
switch

(expression
of
HO
Endonuclease
reporter)

• allow
ma2ng
between
cells

• Red
luciferase
from
one
cell

ac2vate
luciferase

&
HO
Endonuclease


expression
in
another
cell


• one
popula2on
of
yeast
ma2ng
type
switching
in
another
popula2on


Acknowledgements

Thank
you
to:


• TFs
Oliver
Medvedik,
Jenn
Jocz,
and
David


Thompson


• Professors
Alain
Viel,
Jagesh
Shah,
George


Church,

Sarah
Ma8hews,

Pam
Silver,

and
 Tamara
Brenner


• All
of
the
scien2sts
who
generously
gave
us


plasmids
and
reagents,
par2cularly
the
Murray
 Lab

and

the
Sinclair
Lab


• Our
sponsors:
the
Wyss
Ins2tute
at
Harvard
and


HHMI