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
Bacteria to Yeast
Communication
Bacteria (Signal Sender)
Light Yeast
(Means of
(Signal Receiver)
Communication)
Splitting the lac Operon
Spatial Splitting of the
Lac Operon
Derepression
Trans-activating
factor Beta-Gal
production
De-repression of the Operon
The Signal: Bioluminescence from
Luciferase
Luciferase Enzyme + Luciferin + O2  Oxyluciferin + 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
or
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
of
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