Directed
Evolution
of
Acoustic
Reporter
Genes
Using
High-
Throughput
Acoustic
Screening
Robert
C. Hurt,
⊥
Zhiyang
Jin,
⊥
Mohamed
Soufi,
Katie
K. Wong,
Daniel
P. Sawyer,
Hao
K. Shen,
Przemysław
Dutka,
Ramya
Deshpande,
Ruby
Zhang,
David
R. Mittelstein,
and
Mikhail
G. Shapiro
*
Cite
This:
https://doi.org/10.1021/acssynbio.4c00283
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*
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Supporting
Information
ABSTRACT:
A
major
challenge
in
the
fields
of
biological
imaging
and
synthetic
biology
is
noninvasively
visualizing
the
functions
of
natural
and
engineered
cells
inside
opaque
samples
such
as
living
animals.
One
promising
technology
that
addresses
this
limitation
is
ultrasound
(US),
with
its
penetration
depth
of
several
cm
and
spatial
resolution
on
the
order
of
100
μ
m.
Within
the
past
decade,
reporter
genes
for
US
have
been
introduced
and
engineered
to
link
cellular
functions
to
US
signals
via
heterologous
expression
in
commensal
bacteria
and
mammalian
cells.
These
acoustic
reporter
genes
(ARGs)
represent
a
novel
class
of
genetically
encoded
US
contrast
agent,
and
are
based
on
air-filled
protein
nanostructures
called
gas
vesicles
(GVs).
Just
as
the
discovery
of
fluorescent
proteins
was
followed
by
the
improvement
and
diversification
of
their
optical
properties
through
directed
evolution,
here
we
describe
the
evolution
of
GVs
as
acoustic
reporters.
To
accomplish
this
task,
we
establish
high-throughput,
semiautomated
acoustic
screening
of
ARGs
in
bacterial
cultures
and
use
it
to
screen
mutant
libraries
for
variants
with
increased
nonlinear
US
scattering.
Starting
with
scanning
site
saturation
libraries
for
two
homologues
of
the
primary
GV
structural
protein,
GvpA/B,
two
rounds
of
evolution
resulted
in
GV
variants
with
5-
and
14-fold
stronger
acoustic
signals
than
the
parent
proteins.
We
anticipate
that
this
and
similar
approaches
will
help
high-
throughput
protein
engineering
play
as
large
a role
in
the
development
of
acoustic
biomolecules
as
it
has
for
their
fluorescent
counterparts.
KEYWORDS:
gas vesicles,
acoustic
reporter
genes,
ultrasound,
directed
evolution,
high-throughput
screening
■
INTRODUCTION
Acoustic
reporter
genes
(ARGs)
�
genetically
encoded
re-
porters
that
enable
the
imaging
of
gene
expression
using
ultrasound
(US)
�
were
first
introduced
to
bacteria
in
2018
1
and
subsequently
to
mammalian
cells
in
2019.
2
ARGs
are
based
on
genetically
encoded,
gas-filled
protein
nanostructures
called
gas
vesicles
(GVs),
which
evolved
as
intracellular
flotation
devices
allowing
aerophilic
and
photosynthetic
bacteria
to
float
to
oxygenated
and
better-lit
surface
waters.
3,4
GVs
scatter
US
due
to
the
difference
in
the
density
and
compressibility
of
their
gaseous
interior
relative
to
a
surrounding
aqueous
medium.
5
GVs
have
been
the
subject
of
intense
study,
4
−
11
development,
12
and
application
13
−
22
in
recent
years.
23
−
25
ARGs
have
received
considerable
attention
due
to
their
ability
to
enable
noninvasive,
long-term,
real-time
imaging
of
gene
expression
in
both
bacterial
and
mammalian
cells
deep
inside
living
organisms:
in
particular,
ARGs
have
been
used
to
image
tumor
growth
2,12
and
colonization
by
therapeutic
bacteria,
12
protease
activity,
13
phagolysosomal
function,
6
and
intracellular
Ca
2+
dynamics.
7
However,
despite
several
successful
efforts
to
engineer
the
acoustic
and
expression
properties
of
ARGs,
further
improvements
to
the
performance
of
ARGs
are
needed
to
enable
their
most
impactful
applications
�
in
particular,
those
requiring
the
highly
sensitive
and
specific
detection
of
ARGs
expressed
by
bacterial
or
mammalian
cells,
such
as
in
gut
microbiome
or
tumor
imaging.
Unfortunately,
the
methods
currently
available
for
ARG
engineering
and
acoustic
characterization
are
low-throughput,
complex
to
implement,
and
require
a great
deal
of
hands-on
time
per
sample.
In
particular,
manual
loading
and
imaging
of
individual
samples
limits
throughput
to
a handful
of
samples
per
day.
In
contrast,
the
state-of-the-art
high-throughput
methods
used
to
engineer
fluorescent
proteins
can
process
far
larger
libraries
in
shorter
times,
with
less
intervention
from
users:
plate
readers
can
assay
thousands
of
samples
per
run,
and
flow
cytometers
have
been
used
to
screen
libraries
of
10
8
Received:
April
22,
2024
Revised:
June
24,
2024
Accepted:
June
27,
2024
Research
Article
pubs.acs.org/synthbio
© XXXX
The Authors.
Published
by
American
Chemical
Society
A
https://doi.org/10.1021/acssynbio.4c00283
ACS Synth.
Biol.
XXXX,
XXX,
XXX
−
XXX
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mutants
in
a
single
experiment.
8
In
the
past
few
decades,
a
growing
suite
of
protein
engineering
techniques
have
been
developed
9
and
applied
with
remarkable
success
to
improving
fluorescent
proteins,
opsins,
Cas
proteins,
and
other
bio-
Figure
1.
High-throughput
directed
evolution
workflow
for
ARGs.
(A)
Three
regimes
of
GV
response
to
US.
(B)
Roles
of
the
structural
proteins
GvpA/B
and
GvpC
in
GV
structure.
(C)
Diagrams
of
the
gene
clusters
used
as
starting
points
for
evolution.
(D)
Schematic
of
directed
evolution
workflow
for
ARGs.
The
starting
point
GV
structural
protein
is
mutagenized,
then
expressed
in
Escherichia
coli
as
colonies
on
Petri
dishes.
Colonies
that
turn
white
are
assumed
to
produce
GVs,
and
are
picked
and
expressed
in
liquid
culture.
Cultures
of
GV-expressing
E. coli
are
then
loaded
into
agarose
phantoms
and
imaged
using
US
at
15.625
MHz.
The
resulting
nonlinear
US
intensity
data
are
used
to
rank
the
performance
of
mutants
and
select
the
most
promising
ones
for
further
mutagenesis.
(E)
Schematic
of
the
Acoustic
Plate
Reader
(APR),
which
is
used
for
automated
US
image
collection
of
up
to
1152
samples
of
GV-expressing
E. coli
arrayed
in
96-well
agarose
phantoms.
(F)
Image
of
the
graphical
user
interface
for
the
APR.
ACS
Synthetic
Biology
pubs.acs.org/synthbio
Research
Article
https://doi.org/10.1021/acssynbio.4c00283
ACS Synth.
Biol.
XXXX,
XXX,
XXX
−
XXX
B