S
-
1
Supporting Information
Bioorthogonal
L
abeling
E
nables
I
n
S
itu
F
luorescence
I
maging of
E
xpressed
G
as
V
esicle
N
anostructures
Erik Schrunk
1
, Przemys
ł
aw Dutka
1,2,†
, Robert C. Hurt
2
, Di Wu
1,
*, and Mikhail
G.
Shapiro
1,
3
,4,
*
1
Division of Chemistry and
Chemical Engineering, California Institute of Technology; Pasadena, C
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91125,
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2
Division of Biology and Biological Engineering, California Institute of Technology; Pasadena, C
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91125,
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3
Andrew and Peggy Cherng Department of Medical Engineering, California Institute of Technology; Pasadena,
C
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91125, U
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4
Howard Hughes Medical Institute; Pasadena, C
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91125, U
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t
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s
†
Present address: Department of Structural Biology,
Genentech Inc.,
South
San Francisco,
C
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94080,
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t
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*
E
m
a
i
l
:
Di.Wu@caltech.edu
.
P
h
o
n
e
:
(626)
395
-
8560
.
*
E
m
a
i
l
:
mikhail@caltech.edu
.
P
h
o
n
e
:
(
626)
395
-
8588
.
Contents:
Supplementary Figures S1
-
S4
and Supplementa
ry
Methods 1
.
Figure S1: Sequence alignment for
Anabaena flos
-
aquae
gvpA
and
Serratia sp. 39006
gvpA1.
The
two genes have an identity score of 80.6% (58 of 72 amino
acids) and a similarity score of 91.7% (66 of 72 amino
acids).
Colored amino acids represent pairs of amino acids with similar or identical side chains: blue for
hydrophobic
groups, purple for
negatively charged groups
, green for polar
uncharged groups
, red for
positively
charged groups
, orange for glycine, teal for
aromatic groups
(
tyrosine
and histidine)
, and yellow for proline.
S
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2
Figure S2: Opacity screen of mutant gvpA1 plasmids in
E. coli
.
Patches of
E. coli
transformed with
arabinose
-
inducible constructs encoding mutant gvpA1, wild type gvpA1, or GFP were grown on LB agar plates.
Two images, each showing
a set of
four distinct plates, are shown.
The patches
o
n the four plates on the right
represent
duplicate
technical replicates of the corresponding patches
o
n the four plates on the left.
Withi
n each
set of four plates
, t
he two plates on the left
(column 1)
contain the inducer arabinose; the two plates on the right
(column 2)
do not. Each gvpA1 variant (and GFP) is represented by 4 patches arranged into a 2x2 grid in which
each of the 4 patches originates from a different colony from the original transformation (
i.e.
, each
of the
4
patch
es
represents a distinct biological replicate).
2x2 grids of patches are labeled with their corresponding gvpA1 variant
(or GFP) in the column on the left; all other columns are arranged identically.
S
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3
Figure S3: Images of FlAsH
-
labeled GVs in cells transfected with different ratios of
wtGvpA:tcGvpA.
Images of tcGV clusters in fixed HEK 293T cells transfected with 10% (top row) and 25%
(bottom row) tcGvpA. GV clusters are visible under bright
-
field imaging (first column) and are brightly labeled
with FlAsH (second column). The bright
-
field/FlAsH overla
y (third column) demonstrates that the strongest
FlAsH signal overlaps with tcGV clusters. All scale bars 5 μm
.
Cells transfected with
100
% tcGvpA
did not r
esult
in visible GV formation (data not shown).
S
-
4
Figure S4: 3D renderings of tcGV
-
expressing cells
. The cell membrane is depicted in red (Lck
-
mScarlet
-
I), tcGVs in green (FlAsH), and nucleus in blue (DAPI). All scale bars 5
μm.
S
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5
Supplementa
ry
Methods 1:
Estimation
of the GvpA concentration
within a typical
FlAsH imaging voxel
.
To
estimate the molar concentration of GvpA in a typical imaging voxel,
we
first
estimated t
he
number of GvpA
molecules in a typical GV
(
푛
!"#$
)
using
the
length of a typical
Anabaena flos
-
aquae
GV (
퐿
=
500
푛푚
)
(ref. 1)
, the
number of GvpA per helical
turn
(
푛
=
227
)
(ref. 2)
,
and
the
helical
pitch
(
푃
=
4
.
9
푛푚
)
(ref. 2)
using
:
푛
!"#$
=
푛
∗
퐿
/
푃
≈
2
×
10
%
Assuming
a typical imaging vo
xel of
(
500
푛푚
)
&
for FlAsH imaging, a single GV within the voxel
would result in a GvpA concentration of
~
300
μ
푀
. A tightly packed GV cluster
within the voxel
would give a GvpA concentration of
~
10
푚푀
.
(1)
Dutka, P.
,
Metskas, L. A.
,
Hurt, R. C.
,
Salahshoor, H.
,
Wang, T.
-
Y.
,
Malounda, D.
,
Lu, G.
J.
,
Chou, T.
-
F.
,
Shapiro, M. G.
,
Jensen, G. J.
(2023)
Structure of Anabaena Flos
-
Aquae Gas
Vesicles Revealed by Cryo
-
ET. Structure, 31 (5), 518
-
528.e6.
(2)
Huber, S. T.
,
Terwiel, D.
,
Evers, W. H.
,
Maresca, D.
,
Jakobi, A. J.
(2023)
Cryo
-
EM
Structure of Gas Vesicles for Buoyancy
-
Controlled Motility. Cell, 186 (5), 975
-
986.e13.