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

Splitting the lac Operon

Spatial Splitting of the Lac Operon Der erepr epression ession T rans-activ ans-activating ating Beta-Gal Beta-Gal factor actor pr production oduction

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
 Circuit
Diagram
 Array


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 PCB
Concentra2on

 Biodot
Assay
 Assay
 • Immobilized
cells
from
liquid
 • Y190
cells
with
PhyB
DBD/PIF3
 culture
lysed
w/
liquid
nitrogen
 AD
grown
to
10^6
cells/mL
 and
incubated
(30
C)overnight

 • plated
in
100uL
solid
media

 in
150uL
X
gal
buffer
 • PCB
effec2ve
in
inducing
Lac
Z
 • Also
looking
for
B‐Galactosidase
 expression
 ac2vity


Characterization: Laser Based Testing • Pulsed
cells
for
10
sec
 • Same
density
as
PCB
 and
incubated
for
30
 concentra2on
assay
 or
60
min
 • 650
nm
light
pulse
 • 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 
 Poor
batch
quality
of
crude
PCB
 


‐

Control 
 Extract 
 


Experiment
 


+
Control 
 Low
expression
levels
of
red
 


‐

Control 
 luciferase
in
bacteria
(light
 


Experiment
 emission
levels) 
 


+
Control 
 Elevated
B‐gal
expression
level
 


‐

Control 
 of
yeast
cultures
(problems
with
 



Experiment
 induced
background
 expression). 


Successful Bacteria to Yeast Communication?? Experimental:

 Nega=ve
Control:
 Bacterial
Light
+
PCB
 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